Single domain antibody and derivative proteins thereof against CTLA4

ABSTRACT

The present invention relates to the field of medical biology, and discloses a single domain antibody and derivative proteins thereof against CTLA4. In particular, the present invention discloses a CTLA4 binding protein and the use thereof, especially the use for treating and/or preventing CTLA4 relevant diseases such as tumor.

TECHNICAL FIELD

The present invention relates to the field of medical biology, anddiscloses a single domain antibody and derivative proteins thereofagainst CTLA4. In particular, the present invention discloses a CTLA4binding protein and the use thereof, especially the use for treatingand/or preventing CTLA4 relating diseases such as tumor.

BACKGROUND

Animals of Camelidae, such as camels or alpacas, are capable ofproducing a heavy chain antibody that naturally deficient with lightchain. The molecule of heavy chain antibody contains only one heavychain variable region (VHH) and two conventional CH2 and CH3 regions,but has the complete antigen binding function and is not as easy toaggregate as artificially engineered single-chain antibody fragments(scFv). More importantly, the recombinantly expressed VHH domain hasstructural stability and antigen binding activity comparable to that ofthe original heavy chain antibody, and is the smallest unit currentlyknown that can bind to target antigen, called Nanobody or heavy chainsingle domain antibody. Due to its special structural properties, heavychain single domain antibody has the advantages of both traditionalantibodies and small molecule drugs, and overcomes the shortcomings oftraditional antibody, such as long development cycle, low stability, andharsh storage conditions, representing the direction of developing a newgeneration of antibody therapy.

Tumor-associated antigens expressed by tumor cells are the basis forproducing an effective immune response. However, when antigens are boundto major histocompatibility complex (MHC) molecules on the surface ofantigen-presenting cells (APCs) and presented, co-stimulatory signalsare required to promote activation of effector T cells. Studies haveshown that many tumors can escape the patient's own immune system, inpart because of the lack of costimulatory signals to fully activate theT cells, and most likely due to immunosuppression induced by regulatoryT cells (Treg). Binding of CD80 or CD86 on the antigen-presenting cellsto CD28 on the T cells is a key costimulatory signal. Human cytotoxic Tlymphocyte-associated antigen 4 (CTLA4) is a negative regulator of Tcell expression and has a higher affinity for CD80 and CD86. It blocksthe co-stimulatory signal of CD28 and meanwhile activates the T cellnegative regulatory pathway. The immunosuppressive effect of CTLA4 playsan important role in limiting the autoimmune response. However, in thetumor immune response, CTLA4-mediated inhibition mechanism is often oneof the reasons why tumor cells can escape from the immune system. Thus,T cell mediated anti-tumor responses can be enhanced by blocking theinteraction of CTLA4 with CD80 or CD86.

There is still a need in the art for anti-CTLA4 antibody, especially aheavy chain single domain antibody against CTLA4, which can bind toCTLA4 with high affinity and block the binding of CTLA4 to CD80.

BRIEF DESCRIPTION OF THE INVENTION

The present inventors have obtained anti-CTLA4 heavy chain single domainantibody (VHH) with high specificity, high affinity and high stabilityby screening with phage display technology.

In a first aspect, the invention provides a CTLA4-binding proteincomprising an immunoglobulin single variable domain that specificallybinds to CTLA4.

In another aspect, the present invention relates to a nucleic acidmolecule encoding the CTLA4-binding protein, and an expression vectorand host cell containing said nucleic acid molecule.

The present invention further relates to a pharmaceutical compositioncomprising the CTLA4-binding protein of the invention.

The present invention further relates to a method for producing theCTLA4-binding protein of the invention.

The present invention further relates to use of the CTLA4-bindingprotein and pharmaceutical composition of the invention, especially theuse and method for preventing and/or treating CTLA4 relating diseases.

DESCRIPTION OF DRAWINGS

FIG. 1. shows the binding curves of CTLA4 heavy chain single domainantibodies to CTLA4-Fc antigen protein.

FIG. 2. shows the blocking curves of CTLA4 heavy chain single domainantibodies to CD80/CTLA4 interaction.

FIG. 3. shows the blocking curves of CTLA4 single domain antibody-Fcfusion proteins to CD80/CTLA4 interaction.

FIG. 4. shows the sequence alignment of five humanized variants (A) ofantibody No. C27 and four humanized variants (B) of antibody No. C1.

FIG. 5. shows the binding curves of CTLA4 single domain antibody-Fcfusion proteins to CTLA4 (by ELISA).

FIG. 6. shows the binding curves of CTLA4 single domain antibody-Fcfusion proteins to CTLA4 (by ELISA).

FIG. 7. shows the blocking curves of CTLA4 single domain antibody-Fcfusion proteins to CD80/CTLA4 interaction (by competitive ELISA).

FIG. 8. shows the binding curves of tetravalent CTLA4 single domainantibody-Fc fusion proteins to CTLA4 (by competitive ELISA).

FIG. 9. shows the blocking capacity of bivalent or tetravalent CTLA4single domain antibody-Fc fusion proteins to CD80/CTLA4 interaction (bycell neutralization experiment).

FIG. 10. the binding specificity of CTLA4 single domain antibody-Fcfusion proteins to CTLA4 protein detected by flow cytometry.

FIG. 11. shows the binding of tetravalent CTLA4 single domainantibody-Fc fusion proteins to monkey CTLA4 protein detected by flowcytometry.

FIG. 12. shows the activation of PBMC by CTLA4 single domain antibody-Fcfusion proteins.

FIG. 13. shows the comparison of the activation of PBMC by bivalent andtetravalent CTLA4 single domain antibody-Fc fusion proteins.

FIG. 14. shows the activation of CD4+ T cells by CTLA4 single domainantibody-Fc fusion proteins.

FIG. 15. shows that the in vivo inhibition effect of CTLA4 single domainantibody-Fc fusion proteins to MC38 tumor in CTLA4-humanized mice.

FIG. 16. shows change curves of the plasma concentration of bivalent andtetravalent CTLA4 single domain antibody-Fc fusion proteins in rats overtime.

FIG. 17. shows the pharmacokinetic parameters of CTLA4 single domainantibody-Fc fusion proteins in rats.

FIG. 18. shows the heat stability of CTLA4 single domain antibody-Fcfusion proteins.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Unless indicated or defined otherwise, all terms used have their usualmeaning in the art, which will be clear to the skilled person. Referenceis for example made to the standard handbooks, such as Sambrook et al,“Molecular Cloning: A Laboratory Manual” (2nd Ed.), Vols. 1-3, ColdSpring Harbor Laboratory Press (1989); Lewin, “Genes IV”, OxfordUniversity Press, New York, (1990), and Roitt et al., “Immunology” (2ndEd.), Gower Medical Publishing, London, New York (1989), as well as tothe general background art cited herein. Furthermore, unless indicatedotherwise, all methods, steps, techniques and manipulations that are notspecifically described in detail can be performed and have beenperformed in a manner known per se, as will be clear to the skilledperson. Reference is for example again made to the standard handbooks,to the general background art referred to above and to the furtherreferences cited therein.

Unless indicated otherwise, the interchangeable terms “antibody” and“immunoglobulin”—whether used herein to refer to a heavy chain antibodyor to a conventional 4-chain antibody—are used as general terms toinclude both the full-size antibody, the individual chains thereof, aswell as all parts, domains or fragments thereof (including but notlimited to antigen-binding domains or fragments such as VHH domains orVH/VL domains, respectively). In addition, the term “sequence” as usedherein (for example in terms like “immunoglobulin sequence”, “antibodysequence”, “single variable domain sequence”, “VHH sequence” or “proteinsequence”), should generally be understood to include both the relevantamino acid sequence as well as nucleic acid sequences or nucleotidesequences encoding the same, unless the context requires a more limitedinterpretation.

The term “domain” (of a polypeptide or protein) as used herein refers toa folded protein structure which has the ability to retain its tertiarystructure independently of the rest of the protein. Generally, domainsare responsible for discrete functional properties of proteins, and inmany cases may be added, removed or transferred to other proteinswithout loss of function of the remainder of the protein and/or of thedomain.

The term “immunoglobulin domain” as used herein refers to a globularregion of an antibody chain (such as e.g. a chain of a conventional4-chain antibody or of a heavy chain antibody), or to a polypeptide thatessentially consists of such a globular region. Immunoglobulin domainsare characterized in that they retain the immunoglobulin foldcharacteristic of antibody molecules, which consists of a 2-layersandwich of about 7 antiparallel beta-strands arranged in twobeta-sheets, optionally stabilized by a conserved disulfide bond.

The term “immunoglobulin variable domain” as used herein means animmunoglobulin domain essentially consisting of four “framework regions”which are referred to in the art and hereinbelow as “framework region 1”or “FR1”; as “framework region 2” or “FR2”; as “framework region 3” or“FR3”; and as “framework region 4” or “FR4”, respectively; whichframework regions are interrupted by three “complementarity determiningregions” or “CDRs”, which are referred to in the art and hereinbelow as“complementarity determining region” or “CDR1”; as “complementaritydetermining region 2” or “CDR2”; and as “complementarity determiningregion 3” or “CDR3”, respectively. Thus, the general structure orsequence of an immunoglobulin variable domain can be indicated asfollows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. It is the immunoglobulinvariable domain(s) that confer specificity to an antibody for theantigen by carrying the antigen-binding site.

The term “immunoglobulin single variable domain” as used herein means animmunoglobulin variable domain which is capable of specifically bindingto an epitope of the antigen without pairing with an additional variableimmunoglobulin domain. One example of immunoglobulin single variabledomains in the meaning of the present invention is “domain antibody”,such as the immunoglobulin single variable domains VH and VL (VH domainsand VL domains). Another example of immunoglobulin single variabledomains is “VHH domain” (or simply “VHH”) from camelids, as definedhereinafter.

“VHH domains”, also known as heavy chain single domain antibodies, VHHs,V_(H)H domains, VHH antibody fragments, and VHH antibodies, are theantigen binding immunoglobulin variable domain of “heavy chainantibodies” (i.e., “antibodies devoid of light chains”)(Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C,Songa E B, Bendahman N, Hamers R.: “Naturally occurring antibodiesdevoid of light chains”; Nature 363, 446-448 (1993)). The term “VHHdomain” has been used in order to distinguish these variable domainsfrom the heavy chain variable domains that are present in conventional4-chain antibodies (which are referred to herein as “VH domains”) andfrom the light chain variable domains that are present in conventional4-chain antibodies (which are referred to herein as “VL domains”). VHHdomains can specifically bind to an epitope without an additionalantigen binding domain (as opposed to VH or VL domains in a conventional4-chain antibody, in which case the epitope is recognized by a VL domaintogether with a VH domain). VHH domains are small, robust and efficientantigen recognition units formed by a single immunoglobulin domain.

In the context of the present invention, the terms heavy chain singledomain antibody, VHH domain, VHH, V_(H)H domain, VHH antibody fragment,VHH antibody, as well as “Nanobody®” and “Nanobody® domain” (“Nanobody”being a trademark of the company Ablynx N.V.; Ghent; Belgium) are usedinterchangeably.

The amino acid residues of VHH domains from Camelids are numberedaccording to the general numbering for VH domains given by Kabat et al.(“Sequence of proteins of immunological interest”, US Public HealthServices, NIH Bethesda, Md., Publication No. 91), as shown e.g. in FIG.2 of Riechmann and Muyldermans, J. Immunol. Methods 231, 25-38 (1999).According to this numbering,

-   -   FR1 comprises the amino acid residues at positions 1-30,    -   CDR1 comprises the amino acid residues at positions 31-35,    -   FR2 comprises the amino acids at positions 36-49,    -   CDR2 comprises the amino acid residues at positions 50-65,    -   FR3 comprises the amino acid residues at positions 66-94,    -   CDR3 comprises the amino acid residues at positions 95-102, and    -   FR4 comprises the amino acid residues at positions 103-113.

However, it should be noted that—as is well known in the art for VHdomains and for VHH domains—the total number of amino acid residues ineach of the CDRs may vary and may not correspond to the total number ofamino acid residues indicated by the Kabat numbering (that is, one ormore positions according to the Kabat numbering may not be occupied inthe actual sequence, or the actual sequence may contain more amino acidresidues than the number allowed for by the Kabat numbering). This meansthat, generally, the numbering according to Kabat may or may notcorrespond to the actual numbering of the amino acid residues in theactual sequence.

Alternative methods for numbering the amino acid residues of VH domains,which methods can also be applied in an analogous manner to VHH domains,are known in the art. However, in the present description, claims andfigures, the numbering according to Kabat and applied to VHH domains asdescribed above will be followed, unless indicated otherwise.

The total number of amino acid residues in a VHH domain will usually bein the range of from 110 to 120, often between 112 and 115. It shouldhowever be noted that smaller and longer sequences may also be suitablefor the purposes described herein.

Further structural characteristics and functional properties of VHHdomains and polypeptides containing the same can be summarized asfollows:

VHH domains (which have been “designed” by nature to functionally bindto an antigen without the presence of, and without any interaction with,a light chain variable domain) can function as a single, relativelysmall, functional antigen-binding structural unit, domain orpolypeptide. This distinguishes the VHH domains from the VH and VLdomains of conventional 4-chain antibodies, which by themselves aregenerally not suitable for practical application as singleantigen-binding proteins or immunoglobulin single variable domains, butneed to be combined in some form or another to provide a functionalantigen-binding unit (as in for example conventional antibody fragmentssuch as Fab fragments; in scFvs, which consist of a VH domain covalentlylinked to a VL domain).

Because of these unique properties, the use of VHH domains—either aloneor as part of a larger polypeptide—offers a number of significantadvantages over the use of conventional VH and VL domains, scFvs orconventional antibody fragments (such as Fab- or F(ab′)₂-fragments):

-   -   only a single domain is required to bind an antigen with high        affinity and with high selectivity, so that there is no need to        have two separate domains present, nor to assure that these two        domains are present in the right spatial conformation and        configuration (i.e. through the use of especially designed        linkers, as with scFvs);    -   VHH domains can be expressed from a single gene and require no        post-translational folding or modifications;    -   VHH domains can easily be engineered into multivalent and        multispecific formats (formatted);    -   VHH domains are highly soluble and do not have a tendency to        aggregate;    -   VHH domains are highly stable to heat, pH, proteases and other        denaturing agents or conditions and, thus, may be prepared,        stored or transported without the use of refrigeration        equipments, conveying a cost, time and environmental savings;    -   VHH domains are easy and relatively cheap to prepare, even on a        scale required for production;    -   VHH domains are relatively small (approximately 15 kDa, or 10        times smaller than a conventional IgG) compared to conventional        4-chain antibodies and antigen-binding fragments thereof, and        therefore show high(er) penetration into tissues and can be        administered in higher doses than such conventional 4-chain        antibodies and antigen-binding fragments thereof;    -   VHH domains can show so-called cavity-binding properties        (especially due to their extended CDR3 loop, compared to        conventional VH domains) and can therefore also access targets        and epitopes not accessible to conventional 4-chain antibodies        and antigen-binding fragments thereof.

Methods of obtaining VHH domains binding to a specific antigen orepitope have been described earlier, e.g. in WO2006/040153 andWO2006/122786; R. van der Linden et al., Journal of ImmunologicalMethods, 240 (2000) 185-195; Li et al., J Biol Chem., 287 (2012)13713-13721; Deffar et al., African Journal of Biotechnology Vol. 8(12), pp. 2645-2652, 17 Jun. 2009 and WO94/04678.

VHH domains derived from camelids can be “humanized” by replacing one ormore amino acid residues in the amino acid sequence of the original VHHsequence by one or more of the amino acid residues that occur at thecorresponding position(s) in a VH domain from a conventional 4-chainantibody from a human being (also referred to as “sequenceoptimization”, and in addition to humanization, sequence optimizationalso encompasses other modification to the sequence by one or moremutations for providing improved VHH features, such as removingpotential sites for post-translation modification). A humanized VHHdomain can contain one or more fully human framework region sequences,and in a specific embodiment, containing IGHV3 human framework regionsequence.

As used herein, “domain antibodies” especially refer to the VH or VLdomains of non-camelid mammalians, in particular human 4-chainantibodies. In order to bind an epitope as a single antigen bindingdomain, i.e. without being paired with a VL or VH domain, respectively,specific selection for such antigen binding properties is required, e.g.by using libraries of human single VH or VL domain sequences.

Domain antibodies have, like VHHs, a molecular weight of approximately13 to approximately 16 kDa and, if derived from fully human sequences,do not require humanization for e.g. therapeutical use in humans. As inthe case of VHH domains, they are well expressed also in prokaryoticexpression systems, providing a significant reduction in overallmanufacturing cost.

“Domain antibodies” have been described in e.g. Ward, E. S., et al.:“Binding activities of a repertoire of single immunoglobulin variabledomains secreted from Escherichia coli”; Nature 341:544-546 (1989);Holt, L. J. et al.: “Domain antibodies: proteins for therapy”; TRENDS inBiotechnology 21(11):484-490 (2003).

Furthermore, it will also be clear to the skilled person that it ispossible to “graft” one or more of the CDRs mentioned above onto other“scaffolds”, including but not limited to human scaffolds ornon-immunoglobulin scaffolds. Suitable scaffolds and techniques for suchCDR grafting are known in the art.

As used herein, the term “epitope” refers to any antigenic determinanton an antigen to which the paratope of an antibody binds. Antigenicdeterminants typically contain chemically active surface groupings ofmolecules such as amino acids or sugar side chains and typically havespecific three dimensional structural characteristics, as well asspecific charge characteristics. For example, an epitope typicallyincludes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15consecutive or non-consecutive amino acids in a unique spatialconformation, which can be “linear” or “conformational”. See, e.g.,Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G.E. Morris, Ed. (1996). In a linear epitope, all of the points ofinteraction between the protein and the interacting molecule (such as anantibody) occur linearly along the primary amino acid sequence of theprotein. In a conformational epitope, the points of interaction occuracross amino acid residues on the protein that are separated from oneanother.

Epitopes of a given antigen can be identified using a number of epitopemapping techniques, well known in the art. See, e.g., Epitope MappingProtocols in Methods in Molecular Biology, Vol. 66 (Glenn E. Morris,Ed., 1996). For example, linear epitopes may be determined by e.g.,concurrently synthesizing large numbers of peptides on solid supports,the peptides corresponding to portions of the protein molecule, andreacting the peptides with antibodies while the peptides are stillattached to the supports. Such techniques are known in the art anddescribed in, e.g., U.S. Pat. No. 4,708,871; Geysen et al. (1984) Proc.Natl. Acad. Sci. USA 81:3998-4002; Geysen et al. (1986) Molec. Immunol.23:709-715. Similarly, conformational epitopes may be identified bydetermining spatial conformation of amino acids such as by, e.g., x-raycrystallography and 2-dimensional nuclear magnetic resonance. See, e.g.,Epitope Mapping Protocols, supra.

Antibodies can be screened for competitive binding to a same epitope byconventional techniques known in the art. For example, antibodiescompete or cross-compete for binding to antigen can be obtained bycompetitive or cross-competitive assays. A high throughput process forobtaining antibodies binding to a same epitope based upon theircross-competition is described in International Patent Publication No.WO 03/48731. Correspondingly, antibodies and antigen binding fragmentsthereof that compete with the antibody molecules of the invention forbinding to same epitope on CTLA4 can be obtained by conventionaltechniques known in the art.

Generally, the term “specificity” refers to the number of differenttypes of antigens or epitopes to which a particular antigen-bindingmolecule or antigen-binding protein (such as an immunoglobulin singlevariable domain of the invention) can bind. The specificity of anantigen-binding protein can be determined based on its affinity and/oravidity. The affinity, represented by the equilibrium constant for thedissociation of an antigen with an antigen-binding protein (KD), is ameasure for the binding strength between an epitope and anantigen-binding site on the antigen-binding protein: the lesser thevalue of the KD, the stronger the binding strength between an epitopeand the antigen-binding protein (alternatively, the affinity can also beexpressed as the affinity constant (KA), which is 1/KD). As will beclear to the skilled person, affinity can be determined in a mannerknown per se, depending on the specific antigen of interest. Avidity isthe measure of the strength of binding between an antigen-bindingprotein (such as an immunoglobulin, an antibody, an immunoglobulinsingle variable domain or a polypeptide containing it) and the pertinentantigen. Avidity is related to both the affinity between an epitope andits antigen binding site on the antigen-binding protein and the numberof pertinent binding sites present on the antigen-binding protein.

Unless indicated otherwise, the term “CTLA4-binding protein” refers toany protein that can specifically bind to CTLA4. CTLA4-binding proteincan encompass the antibodies or immunoconjugates against CTLA4 asdefined herein. The term “CTLA4-binding protein” encompassesimmunoglobulin super family antibodies (IgSF), or CDR-grafted molecules.

CTLA4-binding molecule of the invention may contain at least oneCTLA4-binding immunoglobulin single variable domain, such as VHH. Insome embodiments, CTLA4-binding molecule of the invention may containtwo, three, four or more CTLA4-binding immunoglobulin single variabledomains such as VHHs. CTLA4-binding protein of the invention may, inaddition to the CTLA4-binding immunoglobulin single variable domains,comprise linkers and/or moieties with effector functions, e.g.half-life-extending moieties like albumin-binding immunoglobulin singlevariable domains, and/or a fusion partner like serum albumin and/or anattached polymer like PEG and/or an Fc region. In some embodiments,CTLA4-binding protein of the invention also encompasses bi-specificantibody, which contains immunoglobulin single variable domains thatbind to different antigens.

Typically, the CTLA4-binding protein of the invention will bind to theantigen (i.e., CTLA4) with a dissociation constant (KD) of preferably10⁻⁷ to 10⁻¹⁰ moles/liter (M), more preferably 10⁻⁸ to 10⁻¹⁰moles/liter, even more preferably 10⁻⁹ to 10⁻¹⁰ moles/liter, or less (asmeasured in a Biacore or in a KinExA or in a Fortibio assay), and/orwith an association constant (KA) of at least 10⁷ M⁻¹, preferably atleast 10⁸M⁻¹, more preferably at least 10⁹ M⁻¹, more preferably at least10¹⁰ M⁻¹. Any KD value greater than 10⁻⁴ M is generally considered toindicate non-specific binding. Specific binding of an antigen-bindingprotein to an antigen or epitope can be determined in any suitablemanner known per se, including, for example, the assays describedherein, Scatchard analysis and/or competitive binding assays, such asradioimmunoassays (RIA), enzyme immunoassays (EIA) and sandwichcompetition assays.

Amino acid residues will be indicated according to the standardthree-letter or one-letter amino acid code, as generally known andagreed upon in the art. When comparing two amino acid sequences, theterm “amino acid difference” refers to insertions, deletions orsubstitutions of the indicated number of amino acid residues at aposition of the reference sequence, compared to a second sequence. Incase of substitution(s), such substitution(s) will preferably beconservative amino acid substitution(s), which means that an amino acidresidue is replaced with another amino acid residue of similar chemicalstructure and which has little or essentially no influence on thefunction, activity or other biological properties of the polypeptide.Such conservative amino acid substitutions are well known in the art,wherein conservative amino acid substitutions preferably aresubstitutions in which one amino acid within the following groups(i)-(v) is substituted by another amino acid residue within the samegroup: (i) small aliphatic, nonpolar or slightly polar residues: Ala,Ser, Thr, Pro and Gly; (ii) polar, negatively charged residues and their(uncharged) amides: Asp, Asn, Glu and Gin; (iii) polar, positivelycharged residues: His, Arg and Lys; (iv) large aliphatic, nonpolarresidues: Met, Leu, He, Val and Cys; and (v) aromatic residues: Phe, Tyrand Trp. Particularly preferred conservative amino acid substitutionsare as follows: Ala into Gly or into Ser; Arg into Lys; Asn into Gln orinto His; Asp into Glu; Cys into Ser; Gln into Asn; Glu into Asp; Glyinto Ala or into Pro; His into Asn or into Gln; Ile into Leu or intoVal; Leu into Ile or into Val; Lys into Arg, into Gln or into Glu; Metinto Leu, into Tyr or into Ile; Phe into Met, into Leu or into Tyr; Serinto Thr; Thr into Ser; Trp into Tyr; Tyr into Trp or into Phe; Val intoIle or into Leu.

“Sequence identity” between two polypeptide sequences indicates thepercentage of amino acids that are identical between the sequences.“Sequence similarity” indicates the percentage of amino acids thateither are identical or that represent conservative amino acidsubstitutions. Methods for evaluating the level of sequence identitybetween amino acid or nucleotide sequences are known in the art. Forexample, sequence analysis softwares are often used to determine theidentity of amino acid sequences. For example, identity can bedetermined by using the BLAST program at NCBI database. Fordetermination of sequence identity, see e.g., Computational MolecularBiology, Lesk, A. M., ed., Oxford University Press, New York, 1988;Biocomputing: Informatics and Genome Projects, Smith, D. W., ed.,Academic Press, New York, 1993; Computer Analysis of Sequence Data, PartI, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey,1994; Sequence Analysis in Molecular Biology, von Heinje, G., AcademicPress, 1987 and Sequence Analysis Primer, Gribskov, M. and Devereux, J.,eds., M Stockton Press, New York, 1991.

A polypeptide or nucleic acid molecule is considered to be “essentiallyisolated”—for example, when compared to its native biological sourceand/or the reaction medium or cultivation medium from which it has beenobtained—when it has been separated from at least one other componentwith which it is usually associated in said source or medium, such asanother protein/polypeptide, another nucleic acid, another biologicalcomponent or macromolecule or at least one contaminant, impurity orminor component. In particular, a polypeptide or nucleic acid moleculeis considered “essentially isolated” when it has been purified at least2-fold, in particular at least 10-fold, more in particular at least100-fold, and up to 1000-fold or more. A polypeptide or nucleic acidmolecule that is “in essentially isolated form” is preferablyessentially homogeneous, as determined using a suitable technique, suchas a suitable chromatographical technique, such as polyacrylamide gelelectrophoresis.

An “affinity-matured” anti-CTLA4 antibody, in particular a VHH or adomain antibody, has one or more alterations in one or more CDRs whichresult in an improved affinity for CTLA4, as compared to the respectiveparent CTLA4-binding molecule. Affinity-matured CTLA4-binding moleculesof the invention may be prepared by methods known in the art, forexample, as described by Marks et al., 1992, Biotechnology 10:779-783,or Barbas, et al., 1994, Proc. Nat. Acad. Sci, USA 91: 3809-3813.; Shieret al., 1995, Gene 169:147-155; Yelton et al., 1995, Immunol. 155:1994-2004; Jackson et al., 1995, J. Immunol. 154(7):3310-9; and Hawkinset al., 1992, J. Mol. Biol. 226(3): 889 896; K S Johnson and R EHawkins, “Affinity maturation of antibodies using phage display”, OxfordUniversity Press 1996.

As used herein, the term “subject” refers to mammalian, especiallyprimate, in particular human.

CTLA4 Binding Protein of the Invention

In a first aspect, the invention provides a CTLA4-binding protein, whichcomprises at least one immunoglobulin single variable domain that canspecifically bind to CTLA4. In some embodiments, said CTLA4-bindingmolecule comprises one immunoglobulin single variable domain thatspecifically binds to CTLA4. In some embodiments, said CTLA4-bindingmolecule comprises two, three, four or more immunoglobulin singlevariable domains that specifically bind to CTLA4. In some embodiments,said CTLA4-binding protein comprises two or more identicalimmunoglobulin single variable domains that specifically bind to CTLA4.In other embodiments, said CTLA4 binding protein comprises two or moredifferent immunoglobulin single variable domains that specifically bindto CTLA4. In some embodiments, said two or more immunoglobulin singlevariable domains that specifically bind to CTLA4 are directly linked toeach other. In some embodiments, said two or more immunoglobulin singlevariable domains that specifically bind to CTLA4 are linked to eachother by a linker. Said linker may be a non-functional amino acidsequence having a length of 1-20 or more amino acids and no secondary orhigher structure. For example, said linker is a flexible linker such asGGGGS, GS, GAP, (GGGGS)×3, and the like.

In some embodiments, said at least one immunoglobulin single variabledomain comprises the CDR1, CDR2 and CDR3 selected from:

(1) CDR1 set forth in SEQ ID NO:1, CDR2 set forth in SEQ ID NO:2, CDR3set forth in SEQ ID NO:3 (corresponding to CDRs of antibody No. 116);

(2) CDR1 set forth in SEQ ID NO:4, CDR2 set forth in SEQ ID NO:5, CDR3set forth in SEQ ID NO:6 (corresponding to CDRs of antibody No. 119);

(3) CDR1 set forth in SEQ ID NO:7, CDR2 set forth in SEQ ID NO:8, CDR3set forth in SEQ ID NO:9 (corresponding to CDRs of antibody No. 128);

(4) CDR1 set forth in SEQ ID NO:10, CDR2 set forth in SEQ ID NO:11, CDR3set forth in SEQ ID NO:12 (corresponding to CDRs of antibody No. 138);

(5) CDR1 set forth in SEQ ID NO:13, CDR2 set forth in SEQ ID NO:14, CDR3set forth in SEQ ID NO:15 (corresponding to CDRs of antibody No. 145);

(6) CDR1 set forth in SEQ ID NO:16, CDR2 set forth in SEQ ID NO:17, CDR3set forth in SEQ ID NO:18 (corresponding to CDRs of antibody No. 155);

(7) CDR1 set forth in SEQ ID NO:19, CDR2 set forth in SEQ ID NO:20, CDR3set forth in SEQ ID NO:21 (corresponding to CDRs of antibody No. 165);

(8) CDR1 set forth in SEQ ID NO:22, CDR2 set forth in SEQ ID NO:23, CDR3set forth in SEQ ID NO:24 (corresponding to CDRs of antibody No. 188);

(9) CDR1 set forth in SEQ ID NO:25, CDR2 set forth in SEQ ID NO:26, CDR3set forth in SEQ ID NO:27 (corresponding to CDRs of antibody No. C1);

(10) CDR1 set forth in SEQ ID NO:28, CDR2 set forth in SEQ ID NO:29,CDR3 set forth in SEQ ID NO:30 (corresponding to CDRs of antibody No.C2);

(11) CDR1 set forth in SEQ ID NO:31, CDR2 set forth in SEQ ID NO:32,CDR3 set forth in SEQ ID NO:33 (corresponding to CDRs of antibody No.C16);

(12) CDR1 set forth in SEQ ID NO:34, CDR2 set forth in SEQ ID NO:35,CDR3 set forth in SEQ ID NO:36 (corresponding to CDRs of antibody No.C22);

(13) CDR1 set forth in SEQ ID NO:37, CDR2 set forth in SEQ ID NO:38,CDR3 set forth in SEQ ID NO:39 (corresponding to CDRs of antibody No.C27);

(14) CDR1 set forth in SEQ ID NO:40, CDR2 set forth in SEQ ID NO:41,CDR3 set forth in SEQ ID NO:42 (corresponding to CDRs of antibody No.C29);

(15) CDR1 set forth in SEQ ID NO:43, CDR2 set forth in SEQ ID NO:44,CDR3 set forth in SEQ ID NO:45 (corresponding to CDRs of antibody No.C38);

(16) CDR1 set forth in SEQ ID NO:46, CDR2 set forth in SEQ ID NO:47,CDR3 set forth in SEQ ID NO:48 (corresponding to CDRs of antibody No.J5);

(17) CDR1 set forth in SEQ ID NO:49, CDR2 set forth in SEQ ID NO:50,CDR3 set forth in SEQ ID NO:51 (corresponding to CDRs of antibody No.J17);

(18) CDR1 set forth in SEQ ID NO:52, CDR2 set forth in SEQ ID NO:53,CDR3 set forth in SEQ ID NO:54 (corresponding to CDRs of antibody No.J29);

(19) CDR1 set forth in SEQ ID NO:55, CDR2 set forth in SEQ ID NO:56,CDR3 set forth in SEQ ID NO:57 (corresponding to CDRs of antibody No.J35);

(20) CDR1 set forth in SEQ ID NO:58, CDR2 set forth in SEQ ID NO:59,CDR3 set forth in SEQ ID NO:60 (corresponding to CDRs of antibody No.J37);

(21) CDR1 set forth in SEQ ID NO:61, CDR2 set forth in SEQ ID NO:62,CDR3 set forth in SEQ ID NO:63 (corresponding to CDRs of antibody No.J38);

(22) CDR1 set forth in SEQ ID NO:64, CDR2 set forth in SEQ ID NO:65,CDR3 set forth in SEQ ID NO:66 (corresponding to CDRs of antibody No.J39);

(23) CDR1 set forth in SEQ ID NO:67, CDR2 set forth in SEQ ID NO:68,CDR3 set forth in SEQ ID NO:69 (corresponding to CDRs of antibody No.J42);

(24) CDR1 set forth in SEQ ID NO:70, CDR2 set forth in SEQ ID NO:71,CDR3 set forth in SEQ ID NO:72 (corresponding to CDRs of antibody No.J69); and

(25) CDR1 set forth in SEQ ID NO:73, CDR2 set forth in SEQ ID NO:74,CDR3 set forth in SEQ ID NO:75 (corresponding to CDRs of antibody No.J78).

In some embodiments, said at least one immunoglobulin single variabledomain of the CTLA4-binding protein of the invention is VHH. In somespecific embodiments, said VHH comprises an amino acid sequence of anyone of SEQ ID NOs:76-100. In some other embodiments, said VHH is ahumanized VHH. Said humanized VHH comprises an amino acid sequencehaving at least 80%, preferably at least 90%, more preferably at least95%, even more preferably at least 99% sequence identity to any one ofSEQ ID NOs: 76-100. Alternatively, the amino acid sequence of said VHHcontains one or more amino acid substitutions, preferably conservativeamino acid substitutions, compared with any one of SEQ ID NOs: 76-100.For example, said VHH contains 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10conservative amino acid substitutions. In some specific embodiments,said humanized VHH comprises an amino acid sequence of any one of SEQ IDNOs:101-109.

In some embodiments, the CTLA4-binding protein of the invention isobtained by affinity maturation. The CTLA4-binding protein obtained byaffinity maturation may have one or more alterations in one or moreCDRs, such alterations result in an increased affinity to CTLA4 whencompared with parent CTLA4-binding protein.

In some embodiments, the CTLA4-binding protein of the invention, inaddition to the at least one immunoglobulin single variable domain thatcan specifically bind to CTLA4, further comprises an immunoglobulin Fcregion. Inclusion of an immunoglobulin Fc region in the CTLA4-bindingprotein of the invention allows the binding protein to form dimmers, andalso allows extension of the in vivo half-life of said binding protein.Fc region that can be used in the invention may be derived fromimmunoglobulins of different subtypes, such as IgG (e.g, IgG1, IgG2,IgG3 or IgG4 subtype), IgA1, IgA2, IgD, IgE or IgM. The immunoglobulinFc region generally includes a hinge region or a portion of the hingeregion, the CH2 region, and the CH3 region of the immunoglobulinconstant region.

In some embodiments, mutations can be introduced into wildtype Fcsequence for altering relevant activities mediated by Fc. Said mutationsinclude, but not limited to, a) mutations altering CDC activity mediatedby Fc; b) mutations altering ADCC activity mediated by Fc; or c)mutations altering in vivo half-life mediated by FcRn. Such mutation aredescribed in Leonard G Presta, Current Opinion in Immunology 2008,20:460-470; Esohe E. Idusogie et al., J Immunol 2000, 164: 4178-4184;RAPHAEL A. CLYNES et al., Nature Medicine, 2000, Volume 6, Number 4:443-446; Paul R. Hinton et al., J Immunol, 2006, 176:346-356. Forexample, Fc-mediated ADCC or CDC activity can be increased or removed,or FcRn Affinity can be enhanced or attenuated by mutating 1, 2, 3, 4,5, 6, 7, 8, 9, or 10 amino acids on the CH2 region. In addition, proteinstability can be increased by mutating 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10amino acids of the hinge region.

In some embodiments, mutations may be introduced into the Fc sequencesuch that the mutated Fc tends to form homo-dimmers or hetero-dimmersmore readily. For example, Ridgway, Presta et al. 1996 and Carter 2001mentioned using the knob-hole model of the spatial interaction of aminoacid side chain groups on Fc contacting interface to allow different Fcmutants to form hetero-dimmers more readily; in addition, CN 102558355Aor CN 103388013A discloses to allow different Fc mutants to formhetero-dimmers more readily (CN 102558355A), or Fcs with same mutationsto form homo-dimmers more readily (CN 103388013A), by changing thecharges of the amino acids on Fc contacting interface which in turnchanges the ionic interaction at the Fc contacting interface.

Preferably, said immunoglobulin Fc region is an Fc region of humanimmunoglobulin, more preferably an Fc region of human IgG1. In somespecific embodiments, the amino acid sequence of the immunoglobulin Fcregion is set forth in SEQ ID NO:132. In some embodiments, theN-terminal EPKSC in SEQ ID NO: 132 can be deleted or mutated to EPKSS orMDPKSS.

In some embodiments, in the CTLA4-binding protein of the invention, theimmunoglobulin single variable domain capable of specifically binding toCTLA4 is linked to the immunoglobulin Fc region via a linker. Saidlinker may be a non-functional amino acid sequence of 1-20 or more aminoacids in length, without secondary or higher structure. For example, thelinker is a flexible joint such as GGGGS, GS, GAP, and the like.

In some embodiments, the CTLA4-binding protein of the inventioncomprises one immunoglobulin single variable domain that specificallybinds to CTLA4, which is linked directly or via a linker to animmunoglobulin Fc region, said immunoglobulin Fc region allows saidCTLA4-binding protein to form a dimeric molecule comprising twoCTLA4-binding domains. Such a CTLA4-binding protein is also referred toas a bivalent CTLA4-binding protein. In some embodiments, the dimmer isa homo-dimmer.

In some embodiments, a CTLA4-binding protein of the invention comprisestwo immunoglobulin single variable domains that specifically bind toCTLA4 and an immunoglobulin Fc region, which are linked directly or viaa linker, said immunoglobulin Fc region allows the CTLA4-binding proteinto form a dimeric molecule comprising four CTLA4-binding domains. Such aCTLA4-binding protein is also referred to as a tetravalent CTLA4-bindingprotein. In some embodiments, the dimmer is a homo-dimmer.

In some preferred embodiments, the CTLA4-binding protein of theinvention comprising an immunoglobulin Fc region comprises an amino acidsequence selected from SEQ ID NO:114-128.

In another aspect, the CTLA4-binding protein of the invention alsoencompasses an anti-CTLA4 antibody molecule that binds to the sameepitope as a VHH consisting of the amino acid sequence of any one of SEQID NOs:76-100.

The CTLA4-binding protein of the invention has at least one of thefollowing features:

(a) binding to human CTLA4 with a KD of less than 1×10⁻⁷ M;

(b) blocking the interaction of CTLA4 with CD80 and/or CD86;

(c) enhancing activation of PBMCs and/or T cells;

(d) inhibiting tumor growth.

The CTLA4-binding protein of the invention may bind to CTLA4 with a KDof less than 1×10⁻⁷ M, preferably less than 1×10⁻⁸ M, more preferablyless than 1×10⁻⁹ M, more preferably less than 1×10⁻¹⁰ M.

In some embodiments, the CTLA4-binding protein of the invention canspecifically bind to human CTLA4 and block the interaction of CTLA4 withCD80, and/or interaction of CTLA4 with CD80 and/or CD86.

The CTLA4-binding protein of the invention can inhibit tumor growth byat least about 10%, preferably at least about 20%, more preferably atleast about 30%, more preferably at least about 40%, more preferably atleast about 50%, more preferably at least about 60%, more preferably atleast about 70%, and more preferably at least about 80%.

Furthermore, the CTLA4-binding protein of the invention is resistant toheat treatment. For example, no significant aggregation or degradationcan be observed after treatment at 40° C. for up to 30 days.

Finally, the CTLA4-binding protein of the invention shows bettertolerance in cynomolgus monkeys. For example, up to 30 mg/kg of theadministered dose, no drug-related adverse reactions can be observed.

Nucleic Acid, Vector and Host Cell

In another aspect, the invention relates to a nucleic acid molecule thatencodes the CTLA4-binding proteins of the invention. The nucleic acid ofthe invention may be RNA, DNA or cDNA. According to one embodiment ofthe invention, the nucleic acid of the invention is in essentiallyisolated form.

The nucleic acid of the invention may also be in the form of, may bepresent in and/or may be part of a vector, such as for example aplasmid, cosmid or YAC. The vector may especially be an expressionvector, i.e. a vector that can provide for expression of theCTLA4-binding protein in vitro and/or in vivo (i.e. in a suitable hostcell, host organism and/or expression system). Such expression vectorgenerally comprises at least one nucleic acid of the invention that isoperably linked to one or more suitable regulatory elements, such aspromoter(s), enhancer(s), terminator(s), and the like. Such elements andtheir selection in view of expression of a specific sequence in aspecific host are common knowledge of the skilled person. Specificexamples of regulatory elements and other elements useful or necessaryfor expressing CTLA4-binding protein of the invention include such aspromoters, enhancers, terminators, integration factors, selectionmarkers, leader sequences, reporter genes, and the like.

The nucleic acids of the invention may be prepared or obtained in amanner known per se (e.g. by automated DNA synthesis and/or recombinantDNA technology), based on the information on the amino acid sequencesfor the polypeptides of the invention given herein, and/or can beisolated from a suitable natural source.

In another aspect, the invention relates to recombination host cellsthat express or are capable of expressing one or more CTLA4-bindingprotein of the invention; and/or that contain a nucleic acid of theinvention. According to a particularly preferred embodiment, said hostcells are bacterial cells; other useful cells are yeast cells, fungalcells or mammalian cells.

Suitable bacterial cells include cells from gram-negative bacterialstrains such as strains of Escherichia coli, Proteus, and Pseudomonas,and gram-positive bacterial strains such as strains of Bacillus,Streptomyces, Staphylococcus, and Lactococcus. Suitable fungal cellinclude cells from species of Trichoderma, Neurospora, and Aspergillus.

Suitable fungal cell include cells from species of Trichoderma,Neurospora, and Aspergillus. Suitable yeast cells include cells fromspecies of Saccharomyces (for example, Saccharomyces cerevisiae),Schizosaccharomyces (for example, Schizosaccharomyces pombe), Pichia(for example, Pichia pastoris and Pichia methanolica), and Hansenula.

Suitable mammalian cells include for example HEK293 cells, CHO cells,BHK cells, HeLa cells, COS cells, and the like.

However, amphibian cells, insect cells, plant cells, and any other cellsused in the art for the expression of heterologous proteins can be usedas well.

The invention further provides methods of manufacturing a CTLA4-bindingprotein of the invention, such methods generally comprise the steps of:

-   -   culturing host cells of the invention under conditions that        allow expression of the CTLA4-binding protein of the invention;        and    -   recovering the CTLA4-binding protein expressed by the host cells        from the culture; and    -   optionally further purifying and/or modifying the CTLA4-binding        protein of the invention.

CTLA4-binding proteins of the invention may be produced in a cell as setout above either intracellullarly (e.g. in the cytosol, in theperiplasma or in inclusion bodies) and then isolated from the host cellsand optionally further purified; or they can be produced extracellularly(e.g. in the medium in which the host cells are cultured) and thenisolated from the culture medium and optionally further purified.

Methods and reagents used for the recombinant production ofpolypeptides, such as specific suitable expression vectors,transformation or transfection methods, selection markers, methods ofinduction of protein expression, culture conditions, and the like, areknown in the art. Similarly, protein isolation and purificationtechniques useful in a method of manufacture of a CTLA4-binding proteinof the invention are well known to the skilled person.

However, the CTLA4-binding proteins of the invention can also beobtained by other methods for production of proteins known in the art,such as, chemical synthesis, including solid phase or liquid phasesynthesis.

Pharmaceutical Compositions

In another aspect, the present invention provides a composition, e.g., apharmaceutical composition, containing one or a combination ofCTLA4-binding protein of the present invention, formulated together witha pharmaceutically acceptable carrier. Such compositions may include oneor a combination of (e.g., two or more different) CTLA4-binding proteinsof the invention. For example, a pharmaceutical composition of theinvention can comprise a combination of antibody molecules that bind todifferent epitopes on the target antigen.

Pharmaceutical compositions of the invention also can be administered incombination therapy, i.e., combined with other agents. For example, thecombination therapy can include a CTLA4-binding protein of the presentinvention combined with at least one other anti-tumor agent. Forexample, CTLA4-binding protein of the invention may be administered incombination with antibodies targeting other tumor-specific antigens.Said antibodies targeting other tumor-specific antigens include, but arenot limited to anti-EGFR antibody, anti-EGFR variant antibody,anti-VEGFa antibody, anti-HER2 antibody, or anti-CMET antibody.Preferably, said antibodies are monoclonal. CTLA4-binding protein of thepresent invention can also be used in combination with other tumorimmunotherapy means or tumor-targeting small molecule drugs. The othertumor immunotherapy means include, but are not limited to, therapeuticantibodies against tumor immunomodulatory molecules such as OX40,PDL1/PD1, CD137, etc., or CAR-T treatment means and the like.

Pharmaceutical composition of the present invention can also be used incombination with other tumor treatment means such as radiotherapy,chemotherapy, surgery, or the like, or be used before or afterradiotherapy, chemotherapy, or surgery.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. Preferably, the carrier is suitable forintravenous, intramuscular, subcutaneous, parenteral, spinal orepidermal administration (e.g., by injection or infusion). Depending onthe route of administration, the active compound, i.e., antibody, orimmunoconjugate, may be coated in a material to protect the compoundfrom the action of acids and other natural conditions that mayinactivate the compound.

The pharmaceutical compounds of the invention may include one or morepharmaceutically acceptable salts. A “pharmaceutically acceptable salt”refers to a salt that retains the desired biological activity of theparent compound and does not impart any undesired toxicological effects(see e.g., Berge, S. M., et al. (1977) J. Pharm. Sci. 66:1-19). Examplesof such salts include acid addition salts and base addition salts. Acidaddition salts include those derived from nontoxic inorganic acids, suchas hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic,phosphorous and the like, as well as from nontoxic organic acids such asaliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoicacids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromaticsulfonic acids and the like. Base addition salts include those derivedfrom alkaline earth metals, such as sodium, potassium, magnesium,calcium and the like, as well as from nontoxic organic amines, such asN,N′-dibenzylethylenediamine, N-methylglucamine, chloroprocaine,choline, diethanolamine, ethylenediamine, procaine and the like.

A pharmaceutical composition of the invention also may include apharmaceutically acceptable anti-oxidant. Examples of pharmaceuticallyacceptable antioxidants include: (1) water soluble antioxidants, such asascorbic acid, cysteine hydrochloride, sodium bisulfate, sodiummetabisulfite, sodium sulfite and the like; (2) oil-solubleantioxidants, such as ascorbyl palmitate, butylated hydroxyanisole(BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate,alpha-tocopherol, and the like; and (3) metal chelating agents, such ascitric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaricacid, phosphoric acid, and the like.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents.

Prevention of presence of microorganisms may be ensured both bysterilization procedures, supra, and by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents which delay absorption such as aluminum monostearate andgelatin.

Pharmaceutically acceptable carriers include sterile aqueous solutionsor dispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. The use of such media andagents for pharmaceutically active substances is known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the pharmaceutical compositions ofthe invention is contemplated. Supplementary active compounds can alsobe incorporated into the compositions.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, liposome, or other ordered structuresuitable to high drug concentration. The carrier can be a solvent ordispersion medium containing, for example, water, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyethylene glycol, andthe like), and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed bysterilization microfiltration. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying (lyophilization) that yield a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

The amount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will vary depending upon thesubject being treated, and the particular mode of administration. Theamount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will generally be that amountof the composition which produces a therapeutic effect. Generally, outof one hundred percent, this amount will range from about 0.01 percentto about ninety-nine percent of active ingredient, preferably from about0.1 percent to about 70 percent, most preferably from about 1 percent toabout 30 percent of active ingredient in combination with apharmaceutically acceptable carrier.

Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. It is especially advantageousto formulate parenteral compositions in dosage unit form for ease ofadministration and uniformity of dosage. Dosage unit form as used hereinrefers to physically discrete units suited as unitary dosages for thesubjects to be treated; each unit contains a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on (a) the unique characteristics of the active compound andthe particular therapeutic effect to be achieved, and (b) thelimitations inherent in the art of compounding such an active compoundfor the treatment of sensitivity in individuals.

For administration of the antibody molecule, the dosage ranges fromabout 0.0001 to 100 mg/kg, and more usually 0.01 to 20 mg/kg, of thesubject body weight. For example dosages can be 0.3 mg/kg body weight, 1mg/kg body weight, 3 mg/kg body weight, 5 mg/kg body weight, 10 mg/kgbody weight, 20 mg/kg body weight or 30 mg/kg body weight or within therange of 1-30 mg/kg. An exemplary treatment regime entailsadministration once per week, once every two weeks, once every threeweeks, once every four weeks, once a month, once every 3 months or onceevery three to 6 months, or with a short administration interval at thebeginning (such as once per week to once every three weeks), and then anextended interval later (such as once a month to once every three to 6months).

Alternatively, antibody can be administered as a sustained releaseformulation, in which case less frequent administration is required.Dosage and frequency vary depending on the half-life of the antibody inthe patient. In general, human antibodies show the longest half-life,followed by humanized antibodies, chimeric antibodies, and nonhumanantibodies. The dosage and frequency of administration can varydepending on whether the treatment is prophylactic or therapeutic. Inprophylactic applications, a relatively low dosage is administered atrelatively infrequent intervals over a long period of time. Somepatients continue to receive treatment for the rest of their lives. Intherapeutic applications, a relatively high dosage at relatively shortintervals is sometimes required until progression of the disease isreduced or terminated, and preferably until the patient shows partial orcomplete amelioration of symptoms of disease. Thereafter, the patientcan be administered a prophylactic regime.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the present invention may be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration, without being toxic to the patient. The selecteddosage level will depend upon a variety of pharmacokinetic factorsincluding the activity of the particular compositions of the presentinvention employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular compositions employed, the age, sex, weight, condition,general health and prior medical history of the patient being treated,and like factors well known in the medical arts.

A “therapeutically effective amount” of a CTLA4-binding protein of theinvention preferably results in a decrease in severity of diseasesymptoms, an increase in frequency and duration of disease symptom-freeperiods, or a prevention of impairment or disability due to the diseaseaffliction. For example, for the treatment of CTLA4 relating tumors, a“therapeutically effective amount” preferably inhibits cell growth ortumor growth by at least about 10%, at least about 20%, more preferablyby at least about 40%, even more preferably by at least about 60%, andstill more preferably by at least about 80% relative to untreatedsubjects. The ability to inhibit tumor growth can be evaluated in ananimal model system predictive of efficacy in human tumors.Alternatively, this property of a composition can be evaluated byexamining the ability of the compound to inhibit cell growth; suchinhibition can be determined in vitro by assays known to the skilledpractitioner. A therapeutically effective amount of a therapeuticcompound can decrease tumor size, or otherwise ameliorate symptoms in asubject, for example, to achieve or prolong the progression-freesurvival of cancer patients, prolong the overall survival of cancerpatients. One of ordinary skill in the art would be able to determinesuch amounts based on such factors as the subject's size, the severityof the subject's symptoms, and the particular composition or route ofadministration selected. Furthermore, one skilled in the art can alsodetermine the effective amount of a CTLA4-binding protein of theinvention by examining the ability to activate T cells in vitro.

A composition of the present invention can be administered via one ormore routes of administration using one or more of a variety of methodsknown in the art. As will be appreciated by the skilled artisan, theroute and/or mode of administration will vary depending upon the desiredresults. Preferred routes of administration for CTLA4-binding proteinsof the invention include intravenous, intramuscular, intradermal,intraperitoneal, subcutaneous, spinal or other parenteral routes ofadministration, for example by injection or infusion. The phrase“parenteral administration” as used herein means modes of administrationother than enteral and topical administration, usually by injection, andincludes, without limitation, intravenous, intramuscular, intraarterial,intrathecal, intracapsular, intraorbital, intracardiac, intradermal,intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal, epidural andintrasternal injection and infusion.

Alternatively, a CTLA4-binding protein of the invention can beadministered via a non-parenteral route, such as a topical, epidermal ormucosal route of administration, for example, intranasally, orally,vaginally, rectally, sublingually or topically.

The active compounds can be prepared with carriers that will protect thecompound against rapid release, such as a controlled releaseformulation, including implants, transdermal patches, andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Manymethods for the preparation of such formulations are patented orgenerally known to those skilled in the art. See, e.g., Sustained andControlled Release Drug Delivery Systems, J. R. Robinson, ed., MarcelDekker, Inc., New York, 1978.

Therapeutic compositions can be administered with medical devices knownin the art. For example, in a preferred embodiment, a therapeuticcomposition of the invention can be administered with a needlelesshypodermic injection device, such as the devices disclosed in U.S. Pat.Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824;or 4,596,556. Examples of well-known implants and modules useful in thepresent invention include: U.S. Pat. No. 4,487,603, which discloses animplantable micro-infusion pump for dispensing medication at acontrolled rate; U.S. Pat. No. 4,486,194, which discloses a therapeuticdevice for administering medicaments through the skin; U.S. Pat. No.4,447,233, which discloses a medication infusion pump for deliveringmedication at a precise infusion rate; U.S. Pat. No. 4,447,224, whichdiscloses a variable flow implantable infusion apparatus for continuousdrug delivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drugdelivery system having multi-chamber compartments; and U.S. Pat. No.4,475,196, which discloses an osmotic drug delivery system. Thesepatents are incorporated herein by reference. Many other such implants,delivery systems, and modules are known to those skilled in the art.

In certain embodiments, the CTLA4-binding proteins of the invention canbe formulated to ensure proper distribution in vivo. For example, theblood-brain barrier (BBB) excludes many highly hydrophilic compounds. Toensure that the therapeutic compounds of the invention cross the BBB (ifdesired), they can be formulated, for example, in liposomes. For methodsof manufacturing liposomes, see, e.g., U.S. Pat. Nos. 4,522,811;5,374,548; and 5,399,331. The liposomes may comprise one or moremoieties which are selectively transported into specific cells ororgans, thus enhance targeted drug delivery (see, e.g., V. V. Ranade(1989) J. Clin. Pharmacol. 29:685). Exemplary targeting moieties includefolate or biotin (see, e.g., U.S. Pat. No. 5,416,016 to Low et al);mannosides (Umezawa et al., (1988) Biochem. Biophys. Res. Commun.153:1038): antibodies (P. G. Bloeman et al. (1995) FEBS Lett. 357:140;M. Owais et al. (1995) Antimicrob. Agents Chemother. 39:180); surfactantprotein A receptor (Briscoe et al. (1995) Am. J. Physiol. 1233: 134);p120 (Schreier et al. (1994) J Biol. Chem. 269:9090): see also K.Keinanen; M. L. Laukkanen (1994) FEBS Lett. 346:123; JJ. Killion; LJ.Fidler (1994) Immunomethods 4:273.

Preventing and Treating of Diseases

In another aspect, the present invention provides the use of the CTLA4binding protein, nucleic acid, host cell and pharmaceutical compositionof the invention for preventing and/or treating CTLA4 relating diseases,as well as the corresponding methods. CTLA4 relating diseases that canbe prevented and/or treated with the CTLA4-binding protein of theinvention are described in detailed as follows.

Cancer

Blocking CTLA4 by CTLA4-binding protein of the invention can enhance theimmune response to cancerous cells in the patient. A CTLA4-bindingprotein of the invention may be used alone to inhibit the growth ofcancerous tumors. Or as described below, a CTLA4-binding protein of theinvention may be used in conjunction with other anti-tumor therapies,for example, in conjunction with other immunogenic agents, standardcancer treatments, or other antibodies molecule.

Accordingly, in one embodiment, the invention provides a method ofpreventing and/or treating cancer in a subject, comprising administeringto the subject a therapeutically effective amount of CTLA4-bindingprotein of the invention so as to inhibit growth of tumor cells in thesubject.

Preferred cancers which may be prevented and/or treated using theCTLA4-binding protein of the invention include cancers typicallyresponsive to immunotherapy. Non-limiting examples of preferred cancersfor treatment include lung cancer, ovarian cancer, colon cancer, rectalcancer, melanoma (e.g., metastatic malignant melanoma), renal cancer,bladder cancer, breast cancer, liver cancer, lymphoma, hematologicalmalignancy, head and neck cancer, glioma, gastric cancer, nasopharyngealcancer, laryngeal cancer, cervical cancer, corpus carcinoma,osteosarcoma. Examples of other cancers that may be treated using themethods of the invention include bone cancer, pancreatic cancer,prostatic cancer, skin cancer, cancer of the head or neck, cutaneous orintraocular malignant melanoma, uterine cancer, cancer of the analregion, testicular cancer, carcinoma of the fallopian tubes, carcinomaof the endometrium, carcinoma of the cervix, carcinoma of the vagina,carcinoma of the vulva, Hodgkin's Disease, non-Hodgkin's lymphoma,cancer of the esophagus, cancer of the small intestine, cancer of theendocrine system, cancer of the thyroid gland, cancer of the parathyroidgland, cancer of the adrenal gland, sarcoma of soft tissue, cancer ofthe urethra, cancer of the penis, chronic or acute leukemia includingacute myeloid leukemia, chronic myeloid leukemia, acute lymphoblasticleukemia, chronic lymphocytic leukemia, solid tumors of childhood,lymphocytic lymphoma, cancer of the bladder, cancer of the kidney orureter, carcinoma of the renal pelvis, neoplasm of the central nervoussystem (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axistumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma,epidermoid cancer, squamous cell cancer, T-cell lymphoma,environmentally induced cancers including those induced by asbestos, andcombinations of said cancers.

Optionally, CTLA4-binding protein of the invention can be combined withan immunogenic agent, such as cancerous cells, purified tumor antigens(including recombinant proteins, peptides, and carbohydrate molecules),cells, and cells transfected with genes encoding immune stimulatingcytokines (He et al. (2004) J. Immunol. 173:4919-28). Non-limitingexamples of tumor vaccines that can be used include peptides of melanomaantigens, such as peptides of gp100, MAGE antigens, Trp-2, MART1 and/ortyrosinase, or tumor cells transfected to express the cytokine GM-CSF.

In humans, some tumors have been shown to be immunogenic such asmelanomas. It is anticipated that by raising the threshold of T cellactivation by CTLA4 blockade with CTLA4-binding protein of theinvention, it is possible to activate tumor responses in the host. CTLA4blockade (such as CTLA4 antibody, e.g., the CTLA4-binding protein of theinvention) is likely to be most effective when combined with avaccination protocol. Many experimental strategies for vaccinationagainst tumors have been devised (see Rosenberg, S., 2000, Developmentof Cancer Vaccines, ASCO Educational Book Spring: 60-62; Logothetis, C,2000, ASCO Educational Book Spring: 300-302; Khayat, D. 2000, ASCOEducational Book Spring: 414-428; Foon, K. 2000, ASCO Educational BookSpring: 730-738; see also Restifo, N. and Sznol, M., Cancer Vaccines,Ch. 61, pp. 3023-3043 in DeVita, V. et al. (eds.), 1997, Cancer:Principles and Practice of Oncology. Fifth Edition). In one of thesestrategies, a vaccine is prepared using autologous or allogeneic tumorcells. These cellular vaccines have been shown to be most effective whenthe tumor cells are transduced to express GM-CSF. GM-CSF has been shownto be a potent activator of antigen presentation for tumor vaccination(Dranoff et al. (1993) Proc. Natl. Acad. Sd U.S.A. 90: 3539-43).

The study of gene expression and large scale gene expression patterns invarious tumors has led to the definition of so-called tumor specificantigens (Rosenberg, S A (1999) Immunity 10: 281-7). In many cases,these tumor specific antigens are differentiation antigens expressed inthe tumors and in the cell from which the tumor arose, for examplemelanocyte antigens gp100, MAGE antigens, and Trp-2. More importantly,many of these antigens can be shown to be the targets of tumor specificT cells found in the host. CTLA4-binding protein of the invention may beused in conjunction with a collection of recombinant proteins and/orpeptides expressed in a tumor in order to generate an immune response tothese proteins. These proteins are normally viewed by the immune systemas self-antigens and are therefore tolerant to them. The tumor antigenmay also include the protein telomerase, which is required for thesynthesis of telomeres of chromosomes and which is expressed in morethan 85% of human cancers and in only a limited number of somatictissues (Kim, N et al. (1994) Science 266: 2011-2013). Tumor antigen mayalso be “neo-antigens” expressed in cancer cells because of somaticmutations that alter protein sequence or create fusion proteins betweentwo unrelated sequences (i.e. bcr-abl in the Philadelphia chromosome).

Other tumor vaccines may include the proteins from viruses implicated inhuman cancers such a Human Papilloma Viruses (HPV), Hepatitis Viruses(HBV and HCV) and Kaposi's Herpes Sarcoma Virus (KHSV). Another form oftumor specific antigen which may be used in conjunction with CTLA4blockade (such as CTLA4 antibody, e.g., CTLA4-binding protein of theinvention) is purified heat shock proteins (HSP) isolated from the tumortissue itself. These heat shock proteins contain fragments of proteinsfrom the tumor cells and these HSPs are highly efficient at delivery toantigen presenting cells for eliciting tumor immunity (Suot, R &Srivastava, P (1995) Science 269:1585-1588; Tamura, Y. et al. (1997)Science 278:117-120).

Dendritic cells (DCs) are potent antigen presenting cells that can beused to prime antigen-specific responses. DCs can be produced ex vivoand loaded with various protein and peptide antigens as well as tumorcell extracts (Nestle, F. et al. (1998) Nature Medicine 4: 328-332). DCsmay also be transduced by genetic means to express these tumor antigensas well. DCs have also been fused directly to tumor cells for thepurposes of immunization (Kugler, A. et al. (2000) Nature Medicine6:332-336). As a method of vaccination, DC immunization may beeffectively combined with CTLA4 blockade (such as CTLA4 antibody, e.g.,CTLA4-binding protein of the invention) to activate more potentanti-tumor responses.

CAR-T (Chimeric Antigen Receptor T-Cell Immunotherapy) is another celltherapy for treating tumors. Chimeric Antigen Receptor T-Cell (CAR-Tcells) are T cells from a patient that have been genetically infectedwith a chimeric protein of an antigen-binding moiety of an antibodyagainst certain tumor antigen coupled with CD3-chain or intracellularportion of FcεRIγ for expressing a chimeric antigen receptor (CAR).Also, co-stimulate signaling sequence may be introduced for increasingcytotoxic activity, proliferation and survival of T cells, and promotingthe release of cytokines. After reprogramming, T cells from the patientexpanded in vitro to produce a large number tumor-specific CAR-T cellswhich are then transfused back into the patient for treating tumor.CTLA4 blocking agents (such as CTLA4 antibodies, e.g., the CTLA4 bindingprotein of the invention) may be used in combination with CAR-T celltherapy for activate stronger anti-tumor response.

CTLA4-binding protein of the invention may also be combined withstandard cancer treatments. CTLA4-binding protein of the invention maybe effectively combined with chemotherapeutic regimes. The scientificrationale behind the combined use of CTLA4-binding protein of theinvention and chemotherapy is that cell death, that is a consequence ofthe cytotoxic action of most chemotherapeutic compounds, should resultin increased levels of tumor antigen in the antigen presentationpathway. Other combination therapies that may result in synergy withCTLA4 blockade through cell death are radiation, surgery, and hormonedeprivation. Each of these protocols creates a source of tumor antigenin the host. Angiogenesis inhibitors may also be combined withCTLA4-binding protein of the invention. Inhibition of angiogenesis leadsto tumor cell death which may feed tumor antigen into host antigenpresentation pathways.

CTLA4 binding protein of the invention can also be used in combinationwith antibodies against other tumor-specific antigens. Said antibodiesagainst other tumor-specific antigens include but are not limited toanti-EGFR antibody, anti-EGFR variant antibody, anti-VEGFa antibody,anti-HER2 antibody, or anti-CMET antibody. Preferably, said antibodiesare monoclonal.

CTLA4-binding protein of the invention can also be used in combinationwith bispecific antibodies that target Fc alpha or Fc gammareceptor-expressing effectors cells to tumor cells (see, e.g., U.S. Pat.Nos. 5,922,845 and 5,837,243). Bispecific antibodies can be used totarget two separate antigens. For example anti-Fc receptor/anti tumorantigen (e.g., Her-2/neu) bispecific antibodies have been used to targetmacrophages to sites of tumor. This targeting may more effectivelyactivate tumor specific responses. The T cell arm of these responseswould be augmented by the use of CTLA4 blockade. Alternatively, antigenmay be delivered directly to DCs by the use of bispecific antibodieswhich bind to tumor antigen and a dendritic cell specific cell surfacemarker.

Tumors evade host immune surveillance by a large variety of mechanisms.Many of these mechanisms may be overcome by the inactivation of proteinswhich are expressed by the tumors and which are immunosuppressive. Theseinclude among others TGF-beta (Kehrl, J. et al. (1986) J. Exp. Med. 163:1037-1050), IL-10 (Howard, M. & O'Garra, A. (1992) Immunology Today 13:198-200), and Fas ligand (Hahne, M. et al. (1996) Science 274:1363-1365). Antibodies to each of these entities may be used incombination with CTLA4-binding protein of the invention to counteractthe effects of the immunosuppressive agent and favor tumor immuneresponses by the host.

Other antibodies which may be used to activate host immuneresponsiveness can be used in combination with anti-CTLA4. Anti-CD40antibodies are able to substitute effectively for T cell helper activity(Ridge, J. et al. (1998) Nature 393: 474-478) and can be used inconjunction with CTLA4-binding protein of the invention. Activatingantibodies to T cell costimulatory molecules such as OX-40 (Weinberg, A.et al. (2000) Immunol 164: 2160-2169), 4-1BB (Melero, I. et al. (1997)Nature Medicine 3: 682-685 (1997), and ICOS (Hutloff, A. et al. (1999)Nature 397: 262-266) as well as antibodies which block the activity ofnegative costimulatory molecules such as CTLA-4 (e.g., U.S. Pat. No.5,811,097) or BTLA (Watanabe, N. et al. (2003) Nat Immunol 4:670-9),B7-H4 (Sica, G L et al. (2003) Immunity 18:849-61) may also provide forincreased levels of T cell activation.

Bone marrow transplantation is currently being used to treat a varietyof tumors of hematopoietic origin. While graft versus host disease is aconsequence of this treatment, therapeutic benefit may be obtained fromgraft vs. tumor responses. CTLA4 blockade can be used to increase theeffectiveness of the donor engrafted tumor specific T cells. There arealso several experimental treatment protocols that involve ex vivoactivation and expansion of antigen specific T cells and adoptivetransfer of these cells into recipients in order to antigen-specific Tcells against tumor (Greenberg, R. & Riddell, S. (1999) Science 285:546-51). These methods may also be used to activate T cell responses toinfectious agents such as CMV. Ex vivo activation in the presence ofCTLA4-binding protein of the invention may be expected to increase thefrequency and activity of the adoptively transferred T cells.

Infectious Diseases

Other methods of the invention are used to treat patients that have beenexposed to particular toxins or pathogens. Accordingly, another aspectof the invention provides a method of preventing or treating aninfectious disease in a subject comprising administering to the subjecta CTLA4-binding protein of the invention, such that the subject istreated for the infectious disease.

Similar to its application to tumors as discussed above, CTLA4 blockadecan be used alone, or as an adjuvant, in combination with vaccines, tostimulate the immune response to pathogens, toxins, and self-antigens.Examples of pathogens for which this therapeutic approach may beparticularly useful, include pathogens for which there is currently noeffective vaccine, or pathogens for which conventional vaccines are lessthan completely effective. These include, but are not limited to HTV,Hepatitis (A, B, & C), Influenza, Herpes, Giardia, Malaria, Leishmania,Staphylococcus aureus, Pseudomonas Aeruginosa. CTLA4 blockade isparticularly useful against established infections by agents such as HIVthat present altered antigens over the course of the infections. Thesenovel epitopes are recognized as foreign at the time of anti-human CTLA4administration, thus provoking a strong T cell response that is notdampened by negative signals through CTLA4.

Some examples of pathogenic viruses causing infections treatable bymethods of the invention include HIV, hepatitis (A, B, or C), herpesvirus (e.g., VZV, HSV-1, HAV-6, HSV-II, and CMV, Epstein Barr virus),adenovirus, influenza virus, flaviviruses, echovirus, rhinovirus,coxsackie virus, cornovirus, respiratory syncytial virus, mumps virus,rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus,HTLV virus, dengue virus, papillomavirus, molluscum virus, poliovirus,rabies virus, JC virus and arboviral encephalitis virus.

Some examples of pathogenic bacteria causing infections treatable bymethods of the invention include chlamydia, rickettsial bacteria,mycobacteria, staphylococci, streptococci, pneumonococci, meningococciand conococci, klebsiella, proteus, serratia, pseudomonas, legionella,diphtheria, salmonella, bacilli, cholera, tetanus, botulism, anthrax,plague, leptospirosis, and Lyme's disease bacteria.

Some examples of pathogenic fungi causing infections treatable bymethods of the invention include Candida (albicans, krusei, glabrata,tropicalis, etc.), Cryptococcus neoformans, Aspergillus (fumigatus,niger, etc.), Genus Mucorales (mucor, absidia, rhizophus), Sporothrixschenkii, Blastomyces dermatitidis, Paracoccidioides brasiliensis,Coccidioides immitis and Histoplasma capsulatum.

Some examples of pathogenic parasites causing infections treatable bymethods of the invention include Entamoeba histolytica, Balantidiumcoli, Naegleriafowleri, Acanthamoeba sp., Giardia lambia,Cryptosporidium sp., Pneumocystis carinii, Plasmodium vivax, Babesiamicroti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani,Toxoplasma gondi, Nippostrongylus brasiliensis.

In all of the above methods, CTLA4 blockade can be combined with otherforms of immunotherapy such as cytokine treatment (e.g., interferons,GM-CSF, G-CSF, IL-2), or bispecific antibody therapy, which provides forenhanced presentation of tumor antigens (see, e.g., Holliger (1993)Proc. Natl. Acad. Sci USA 90:6444-6448; Poljak (1994) Structure2:1121-1123).

EXAMPLES

The present invention is further illustrated by the following examples,but the scope of the invention should not be limited to the specificexamples in any way.

Example 1: Screen of Heavy Chain Single Domain Antibody Against CTLA4

1.1 Library Construction and Screening

CTLA4-Fc fusion protein (SEQ ID NO:129) for immunization was expressedby HEK293 cells (pCDNA4, Invitrogen, Cat V86220), purified by Protein Aaffinity chromatography. One Camelus bactrianus was chosen forimmunization. After 4 immunizations, lymphocytes were isolated from 100ml camel peripheral blood, and total RNA was extracted by RNA Extractionkit (QIAGEN). Extracted RNA was reverse transcribed into cDNA usingSuper-Script III FIRST STRANDSUPERMIX kit according to instructions.Nucleic acid fragments encoding heavy chain antibodies were amplified bynested PCR:

First Round PCR:

(SEQ ID NO: 110) Upstream primer: GTCCTGGCTGCTCTTCTACAAGGC;(SEQ ID NO: 111) Downstream primer: GGTACGTGCTGTTGAACTGTTCC.

Second Round PCR:

PCR products from first round PCR as template,

Upstream primer: (SEQ ID NO: 112) GATGTGCAGCTGCAGGAGTCTGGRGGAGG;Downstream primer: (SEQ ID NO: 113) GGACTAGTGCGGCCGCTGGAGACGGTGACCTGGGT.

Target heavy chain single domain antibody nucleic acid fragments wererecovered and cloned into phage display vector pCDisplay-3 (CreativeBiolabs, Cat: VPT4023) using endonuclease PstI and NotI (from NEB). Theproducts were then electroporated into E. coli competent cell TG1, andphage display library for heavy chain single domain antibodies againstCTLA4 was constructed and verified. By plating serial dilutions, librarycapacity was determined as about 10⁸. To determine the insertion ratioof the library, 50 clones were randomly selected for colony PCR. Theresults revealed an insertion ratio of more than 99%.

1.2 Panning for Heavy Chain Single Domain Antibody Against CTLA4

Multi-well plates were coated with CTLA4-Fc fusion protein and Fcprotein at 5 μg/well, 4° C. overnight. On next day, after blocking with1% skim milk at room temperature for 2 hours, 100 μl phages (8×10¹¹ tfu,from the phage display library for camel heavy chain single domainantibodies constructed in 1.1) were added, room temperature for 1 hour.Thereafter, the Fc-unbound phage was again transferred into the wellscoated with CTLA4-Fc fusion protein by washing with PBST (0.05% tween 20in PBS), room temperature for 1 hour. Phages that specifically bind toCTLA4 were dissociated with triethylammonium (100 mM), and used toinfect E. coli TG1 in log phase, producing phages which were thenpurified for next round screen. The same screen was repeated for 3-4rounds. Thereby, positive clones were enriched, achieving the purpose ofselecting CTLA4 specific antibodies from the antibody library by phagedisplay technology.

1.3 Specific Selection of Individual Positive Clones by PhageEnzyme-Linked Immunoassay (ELISA)

CTLA4 binding positive phages obtained after 3 rounds of panning wereused to infect blank E. coli and plated. 96 single colonies wererandomly selected for culturing, and phages were produced and purifiedrespectively. Plates were coated with CTLA4-Fc fusion protein at 4° C.overnight; sample phages as obtained were added (blank phages ascontrol) and incubated at room temperature for 1 hour. Primary antibody,mouse anti-HA tag antibody (Beijing Kangwei Shiji Biotech. Ltd.), wasadded after washes and incubated at room temperature, 1 hour forreaction. Secondary antibody, goat anti-mouse alkaline phosphataselabeled antibody (Amyject Scientific Ltd.) was added after washes andincubated at room temperature, 1 hour for reaction. Alkaline phosphatasechromogenic solution was added after washes, and absorption value wasread at 405 nm wave length. When the OD of the sample well is 3 timeshigher than the OD of control well, the sample is determined aspositive. Bacteria in the positive wells were transferred to andcultured in LB liquid medium supplemented with 100 μg/ml Ampicillin forplasmid extraction and subsequent sequencing.

The protein sequences of each clone were analyzed according to thesequence alignment software Vector NTI. Clones with the same CDR1, CDR2,and CDR3 sequences are considered as the same antibody, while cloneswith different CDR sequences are considered as different antibody, andthose early terminated sequences were excluded. A total of 34 differentantibodies were finally obtained.

Example 2 Preliminary Evaluation of Heavy Chain Single Domain AntibodiesAgainst CTLA4

2.1 Expression of Heavy Chain Single Domain Antibodies in E. coli andPurification Thereof

The coding sequences of the 34 heavy chain single domain antibodiesobtained by sequencing analysis were subcloned into the expressionvector PET32b (Novagen, product number: 69016-3) and the correctrecombinant plasmid was transformed into expression host strain BL1(DE3) (Tiangen Biotech, CB105-02), plated on LB solid medium containing100 micrograms per milliliter ampicillin overnight at 37° C. Singlecolonies were inoculated and cultured overnight, transferred in the nextday for expansion at 37° C. by shaking. When the culture reached ODvalue of 0.6-1, 0.5 mM IPTG was added for induction, 28° C. overnightwith shaking. The next day, the bacteria were harvested bycentrifugation, and lysed to obtain antibody crude extracts. Nickel ionaffinity chromatography was then used to purify the antibody proteins,resulting in antibody proteins of more than 90% purity.

2.2 Specific Binding of the Candidate CTLA4 Heavy Chain Single DomainAntibody to Human CTLA4 Protein

Plates were coated with CTLA4-Fc fusion protein overnight at 4° C. and10 ng of the heavy chain single-domain antibody obtained in Example 2.1(the control was a single domain antibody not binding to the CTLA4-Fcprotein) was added to each well and allowed to react for 1 hour at roomtemperature. After washing, primary antibody anti-His tag antibody(purchased from Beijing Kangwei Century Biotechnology Co., Ltd.) wasadded and reacted for 1 hour at room temperature. After washing, asecondary goat anti-mouse horseradish peroxidase-labeled antibody(Yiqiao Shenzhou, Cat: SSA007200) was added and reacted for 1 hour atroom temperature. After washing, chromogenic agent was added and theabsorbance was read at 405 nm.

Plates were coated with Fc protein overnight at 4° C. and 10 ng of theheavy chain single domain antibody obtained in Example 2.1 was added toeach well (control was a single domain antibody against other unrelatedtargets) and allowed to react for 1 hour at room temperature. Afterwashing, an rabbit anti-human Fc antibody (purchased from Shanghai PuXin Biotechnology Co., Ltd.) was added and reacted for 1 hour at roomtemperature. After washing, goat anti-rabbit horseradish peroxidaselabeled antibody (purchased from Shanghai Pu Xin Biotechnology Co.,Ltd.) was added and reacted at room temperature for 1 hour. Afterwashing, chromogenic agent was added and the absorbance was read at 405nm.

The candidate antibody is considered as binding to the CTLA4-Fc proteinwhen the ratio of the OD value for the CTLA4-Fc protein divided by theOD value for the blank control is greater than or equal to 4; andsimultaneously, the above antibody capable of binding to CTLA4-Fcantigen protein, when the ratio of the OD value for binding to CTLA4-Fcdivided by the OD value for binding Fc protein is >=5, is considered asspecifically binding to the CTLA4 moiety rather than the Fc moiety.

The results showed that out of the 34 antibodies, 25 (bold in bold)could specifically bind to CTLA4 without binding to Fc. The results areshown in the following Table 1:

TABLE 1 Antibody OD (against OD (against ODa/OD SEQ No. CTLA4) Fc)ODa/ODb blank ID NO C1 1.201 0.053 42.9 22.7 84 C2 1.231 0.035 44.0 35.285 C16 1.786 0.053 63.8 33.7 86 C22 0.848 0.06 30.3 14.1 87 C23 0.9511.057 34.0 0.9 C24 0.231 0.114 8.3 2.0 C25 0.091 0.03 3.3 3.0 C27 1.310.049 46.8 26.7 88 C29 1.513 0.085 54.0 17.8 89 C34 1.022 0.945 36.5 1.1C38 1.022 0.045 36.5 22.7 90 C46 0.621 0.241 22.2 2.6 J5 1.21 0.036 43.233.6 91 J17 1.4 0.046 50.0 30.4 92 J24 0.032 0.041 1.1 0.8 J29 1.4390.036 51.4 40.0 93 J34 0.932 1.023 33.3 0.9 J35 0.823 0.036 29.4 22.9 94J37 1.201 0.034 42.9 35.3 95 J38 1.031 0.752 36.8 1.4 96 J39 1.747 0.04262.4 41.6 97 J41 0.055 0.064 2.0 0.9 J42 1.086 0.045 38.8 24.1 98 J470.062 0.041 2.2 1.5 J69 0.635 0.037 22.7 17.2 99 J78 0.632 0.121 22.65.2 100 116 0.234 0.046 8.4 5.1 76 119 0.537 0.037 19.2 14.5 77 1281.268 0.036 45.3 35.2 78 138 1.843 0.041 65.8 45.0 79 145 1.487 0.06553.1 22.9 80 155 1.724 0.051 61.6 33.8 81 165 1.214 0.051 43.4 23.8 82188 0.945 1.021 33.8 0.9 83 Blank 0.028 0.035 0.8 0.82.3 Examination of the Blocking Effect of CTLA4 Heavy ChainSingle-Domain Antibody to the Interaction Between CD80 and CTLA4 byCompetitive ELISA

CTLA4-Fc protein and CD80-Fc protein (SEQ ID NO: 130) were obtained byexpression in HEK293 cells (pCDNA4, Invitrogen, Cat V86220).Biotinylated protein CD80-Fc-Biotin was obtained using the ThermoBiotinlytion kit.

The plates were coated overnight at 4° C. with CTLA4-Fc fusion proteinat 0.5 μg/well followed by addition for each well of 500 ng of the heavychain single domain antibody that specifically binds to CTLA4 confirmedin Example 2.2 (controls are single domain antibodies against otherunrelated targets or simply buffer) and 25 ng of CD80-Fc-Biotin (noantibody or protein was added to the blank group, only an equal volumeof buffer was added), and allowed to react for 1 hour at roomtemperature. SA-HRP (purchased from Sigma) was added and allowed toreact for 1 hour at room temperature. After adding chromogenic solution,absorbance was read at 405 nm wavelength. When the sample OD valuerelative to the control OD value is <0.8, the antibody is considered aspossessing blocking effect.

As shown in Table 2, the antibody No. C1, C16, C27, J37, J42, 128, 145,155, 165 showed a blocking effect on the CD80/CTLA4 interaction.

TABLE 2 Sample OD Control 1 2.453 Control 2 2.391 blank 0.016 C1 0.054C16 1.893 C2 2.517 C22 2.474 C27 0.04 C29 2.431 C38 2.284 J17 2.528 J292.502 J35 2.57 J37 1.061 J38 2.357 J39 2.133 J42 0.94 J5 2.504 J69 2.343J78 2.413 116 2.579 119 2.534 128 0.893 138 1.918 145 0.597 155 0.746165 1.838 188 2.6332.4 Binding of CTLA4 Heavy Chain Single Domain Antibody to Mouse CTLA4Protein

Mouse CTLA4-Fc protein (SEQ ID NO: 131) was obtained by expression inHEK293 cells (pCDNA4, Invitrogen, Cat V86220). Biotinylated proteinmCTLA4-Fc-Biotin was obtained using the Thermo Biotinlytion kit.

The plates were coated with the heavy chain single domain antibodyobtained in Example 2.1 (control group is a single domain antibodyagainst other unrelated target), that is, four antibodies 145, 155, C1and C27 with better blocking effect selected according to the results ofExample 2.3, at 0.5 μg/well overnight at 4° C., and 100 μg of mouseCTLA4-Fc fusion protein was added to each well and reacted for 1.5 hoursat room temperature. Thereafter, SA-HRP (purchased from Sigma) wasadded, and the mixture was reacted at room temperature for 1.5 hours.After washing, chromogenic agent was added and the absorbance was readat 405 nm. The results are shown in Table 3.

TABLE 3 Antibody No. OD (against mouse CTLA4) 145 0.026 155 0.032 C10.042 C27 0.021 Control 0.026 blank 0.027

It can be seen that the heavy chain single domain antibody of humanCTLA4 of the present invention does not bind to the mouse CTLA4-Fcprotein.

2.5 Binding Curves of CTLA4 Heavy Chain Single Domain Antibodies toCTLA4-Fc Antigen Protein

The plates were coated with the obtained CTLA4 heavy chain single-domainantibody at 0.5 μg/well overnight at 4° C. followed by addition of agradient dilution series of CTLA4-Fc fusion protein and allowed to reactfor 1 hour at room temperature. After washing, goat anti-human IgG-Fchorseradish peroxidase labeled antibody (lakepharma) was added andallowed to react for 1 hour at room temperature. After washing,horseradish peroxidase c chromogenic solution was added and theabsorbance was read at a wavelength of 405 nm. SotfMax Pro v5.4 was usedfor data processing and mapping analysis to get binding curve of theantibody to CTLA4 and EC50 value (for antibody No. 14, about 50 ng/ml;for antibody No. 155, about 13 ng/ml; for antibody No. C1, about 123ng/ml; for antibody No. C27, about 93 ng/ml) through four-parameterfitting. The results of 145 and 155 are shown in FIG. 1A, the results ofC1 and C27 are shown in FIG. 1B.

2.6 Blocking Curves of CTLA4 Heavy Chain Single-Domain Antibodies on theInteraction Between CD80 and CTLA4

Plates were coated with 0.5 μg/well CTLA4-Fc fusion protein overnight at4° C. followed by the addition of 100 uL of a gradient dilution series(containing 250 ng/mL CD80-Fc-Biotin) of 100 μL CTLA4 blocking singledomain antibody obtained in Example 2.1 per well, allowed to react for 1hour at room temperature. SA-HRP (purchased from Sigma) was added andallowed to react for 1 hour at room temperature. After addingchromogenic solution, absorbance was read at 405 nm wavelength.

SotfMax Pro v5.4 was used for data processing and graphical analysis toobtain blocking curve and IC50 value of antibody No. C1, C27, 145, 155to CD80/CTLA4 through four-parameter fitting (IC50 for antibody No. C1is 852 ng/mL, for antibody No. C27 is approximately 731 ng/ml, forantibody No. 145 is approximately 1.947 μg/ml, for antibody No. 155 isapproximately 4.690 μg/ml). The results of 145 and 155 are shown in FIG.2A, the results of C1 and C27 are shown in FIG. 2B.

2.7 Preparation of Fc Fusion Protein of CTLA4 Single Domain Antibody

The amino acid sequence of human IgG1-Fc region (SEQ ID NO: 132) wasobtained based on the constant region amino acid sequence of humanimmunoglobulin gamma 1 (IgG1) from the protein database Uniprot(P01857). The nucleic acid fragment encoding human IgG1-Fc was obtainedfrom human PBMC total RNA by reverse transcription PCR, and the nucleicacid fragment encoding the fusion protein of CTLA4 single domainantibody obtained in the above Example and Fc was obtained byoverlapping PCR, then subcloned into vector pCDNA4 (Invitrogen, CatV86220).

Recombinant single domain antibody-Fc fusion protein plasmid wastransfected into HEK293 cells for antibody expression. The recombinantexpression plasmids were diluted with Freestyle 293 medium and addedinto PEI (polyethylenimine) solution for transformation. Eachplasmid/PEI mixture was added to HEK293 cell suspension and incubated at37° C. and 10% CO₂ at 90 rpm. At the same time, 50 μg/L IGF-1 was added.Four hours later EX293 medium, 2 mM glutamine and 50n/L IGF-1 weresupplemented, cultured at 135 rpm. After 24 hours, 3.8 mM VPA was added.After cultured for 5 to 6 days, the transient expression supernatant wascollected and purified by Protein A affinity chromatography to obtainthe target CTLA4 single domain antibody-Fc fusion protein.

The sequences of Fc fusion proteins of antibodies No. C1, C27, 145, 155are shown in SEQ ID NO: 114-117, respectively.

2.8 Blocking Curves of CTLA4 Heavy Chain Single-Domain Antibody FcFusion Proteins on the Interaction of CD80 and CTLA4

Plates were coated with CTLA4-Fc fusion protein 0.5 μg/well overnight at4° C. followed by the addition of 100 μL of a gradient dilution series(containing 250 ng/mL CD80-Fc-Biotin) of CTLA4 blocking single domainantibody obtained in Example 2.7 per well, allowed to react for 1.5 hourat room temperature. SA-HRP (purchased from Sigma) was added and allowedto react for 1.5 hour at room temperature. After adding chromogenicsolution, absorbance was read at 405 nm wavelength.

SotfMax Pro v5.4 was used for data processing and graphical analysis toobtain blocking curve and IC50 value of antibody No. 145, 155 toCD80/CTLA4 through four-parameter fitting (IC50 for antibody No. 145 isapproximately 339 ng/ml, for antibody No. 155 is approximately 261ng/ml). The results are shown in FIG. 3.

Example 3 Humanization of CTLA4 Single Domain Antibodies

The humanization is performed by the method of protein surface aminoacid humanization (resurfacing) and universal framework grafting methodfor VHH humanization (CDR grafting to a universal framework).

The steps of humanization are as follows: The homologous modeling ofantibody No. C27 and C1 were performed with the modeling softwareModeller9. The reference homologous sequence is cAb-Lys3 antibody (PDBcode: 1XFP), and the relative solvent accessibility of the amino acidsis calculated according to the three-dimensional structure of theprotein. If one of the amino acids of antibody No. C27 and C1 areexposed to a solvent, it was replaced with the amino acid at the sameposition of the reference human antibody DP-47 sequence, until allsubstitutions were completed.

The antibody No. C27 was humanized, and five humanized variants of theantibody No. C27 were obtained; the antibody No. C1 was humanized, andfour humanized variants of the antibody No. C1 were obtained. Table 4lists the SEQ ID No of these humanized variants as well as the aminoacid changes therein, with amino acid residue numbers following Kabatnumbering. FIG. 4a shows the alignment of C27 humanized sequences, FIG.4b shows the alignment of C1 humanized sequences.

TABLE 4 SEQ ID NO Q5V S11L A14P G19R E44G R45L V46E Q71R I73N A74S K83RP84A M89V S91Y C72V1 101 ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ C27V2 102 ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓C27V3 103 ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ C27V4 104 ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ C27V5105 ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ C1V1 106 ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ C1V2 107 ✓ ✓ ✓ ✓✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ C1V3 108 ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ C1V4 109 ✓ ✓ ✓ ✓ ✓ ✓✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓

Example 4 Preparation of CTLA4 Blocking Antibody Protein Using MammalianCells

4.1 Preparation of Fc Fusion Protein of CTLA4 Single Domain Antibody

The amino acid sequence of human IgG1-Fc region (SEQ ID NO: 132) wasobtained based on the constant region amino acid sequence of humanimmunoglobulin gamma1 (IgG1) from the protein database Uniprot (P01857).The nucleic acid fragment encoding human IgG1-Fc was obtained from humanPBMC total RNA by reverse transcription PCR, and the nucleic acidfragment encoding the fusion protein of CTLA4 single domain antibodyobtained in the above Example and Fc was obtained by overlapping PCR,then subcloned into vector pCDNA4 (Invitrogen, Cat V86220).

Recombinant single domain antibody-Fc fusion protein plasmid wastransfected into HEK293 cells for antibody expression. The recombinantexpression plasmids were diluted with Freestyle 293 medium and addedinto PEI (polyethylenimine) solution for transformation. Eachplasmid/PEI mixture was added to HEK293 cell suspension and incubated at37° C. and 10% CO₂ at 90 rpm. At the same time, 50 μg/L IGF-1 was added.Four hours later EX293 medium, 2 mM glutamine and 50 μg/L IGF-1 weresupplemented, cultured at 135 rpm. After 24 hours, 3.8 mM VPA was added.After cultured for 5 to 6 days, the transient expression supernatant wascollected and purified by Protein A affinity chromatography to obtainthe target CTLA4 single domain antibody-Fc fusion protein.

The sequences of the CTLA4 single domain antibody-Fc fusion proteins asobtained are shown in SEQ ID NO: 114-126, respectively, wherein SEQ IDNO: 118-126 are Fc fusion proteins of humanized CTLA4 single domainantibodies. These fusion proteins comprise one CTLA4 binding domain andform homo-dimmers, each of which comprises two CTLA4 binding domains,such that these fusion proteins are also referred to as bivalent CTLA4single domain antibody-Fc fusion protein.

4.2 Preparation of CTLA4 Antibodies from BMS

The gene of anti-CTLA4 antibody ipilimumab from BMS, Inc. was cloned bythe method for antibody 10D1 in US20020086041 and cloned into the vectorpCDNA4.

The recombinant plasmid was transiently transfected into HEK293 cells bythe same method as in Example 4.1, and the resulting anti-CTLA4 antibodyof BMS was renamed as 10D1.

4.3 Comparison of the Expression of CTLA4 Single-Domain Antibody FcFusion Protein and the Known CTLA4 Antibodies

Using the same expression system and transient transfection conditions,the expression level of the CTLA4 single-domain antibody Fc fusionprotein of the present invention was higher than 400 mg/L, while theexpression level of the antibody 10D1 was about 150 mg/L. This resultindicates that the CTLA4 single-domain antibody Fc fusion protein of thepresent invention is more stable in structure and can result in higherexpression level than the known CTLA4 antibodies.

4.4 Preparation of Tetravalent CTLA4 Single Domain Antibody Fc FusionProtein

A nuclei acid fragment encoding a fusion protein of two tandem CTLA4single domain antibodies and one Fc region was obtained by overlappingPCR, and then subcloned into vector pCDNA4 (Invitrogen, Cat V86220). Theconstructed recombinant plasmid was used to transfect HEK293 cells fortransient expression by the same method as described in 4.1. A fusionprotein of two tandem CTLA4 single domain antibodies and one Fc wasobtained, which could form a dimeric molecule comprising fourCTLA4-binding domains, also called tetravalent CTLA4 single domainantibody Fc fusion protein.

The mean transient expression level of the tetravalent CTLA4 singledomain antibody Fc fusion proteins is about 400 mg/L, comparable to thatof the bivalent CTLA4 single domain antibody Fc fusion proteins.

Example 5 Characterization of CTLA4 Single-Domain Antibody Fc FusionProtein

5.1 Binding Ability of CTLA4 Single Domain Antibody Fc Fusion Protein toCTLA4 (by ELISA)

Plates were coated with 0.5 μg/well CTLA4 single-domain Fc fusionprotein obtained by Example 2.7 and 4.1 or 10D1 protein obtained byExample 4.2 overnight at 4° C. followed by the addition of a gradientdilution series of CTLA4-Fc-Biotin, allowed to react for 1 hour at roomtemperature. SA-HRP (purchased from Sigma) was added and allowed toreact for 1.5 hour at room temperature. After adding chromogenicsolution, absorbance was read at 405 nm wavelength.

SotfMax Pro v5.4 was used for data processing and graphical analysis.Through four-parameter fitting, binding curve and EC50 value of theantibody to CTLA4 (all test antibody EC50 value of about 60-70 ng/mL)were obtained to reflect the affinity to CTLA4.

The results are shown in FIG. 5, where the longitudinal coordinate isOD405 and the horizontal ordinate is the concentration of CTLA4 singledomain antibody Fc-fusion protein (or 10D1 protein) (in ng/mL); fourdifferent humanized forms of the Fc fusion protein of antibody No. C27were labeled as: huC27v1-LdFc (SEQ ID NO:118), huC27v2-LdFc (SEQ IDNO:119), huC27v3-LdFc (SEQ ID NO:120), huC27v4-LdFc (SEQ ID NO:121). Thefour proteins have comparable affinity for CTLA4, and comparable tounhumanized C27-LdFc and known CTLA4 antibody 10D1 of BMS.

5.2 Binding Ability of CTLA4 Single-Domain Antibody Fc Fusion Protein toCTLA4 (by ELISA)

Plates were coated with 0.5 μg/well CTLA4 single-domain Fc fusionprotein obtained by Example 2.7 and 4.1 overnight at 4° C., followed bythe addition of a gradient dilution series of CTLA4-Fc-Biotin, allowedto react for 1 hour at room temperature. SA-HRP (purchased from Sigma)was added and allowed to react for 1.5 hour at room temperature. Afteradding chromogenic solution, absorbance was read at 405 nm wavelength.

SotfMax Pro v5.4 was used for data processing and graphical analysis.Through four-parameter fitting, binding curve and EC50 value of theantibody to CTLA4 (all test antibody EC50 value of about 25-35 ng/mL)were obtained to reflect the affinity to CTLA4.

The results are shown in FIG. 6, where the longitudinal coordinate isOD405 and the horizontal ordinate is the concentration of CTLA4 singledomain antibody Fc-fusion protein (in ng/mL); triangle represents thehumanized form Fc fusion protein of the antibody No. C27, huC27v3-LdFc(SEQ ID NO: 120), square represents the humanized form Fc fusion proteinof the antibody No. C1, huC1v4-Ld-Fc (SEQ ID NO: 126), circle representsthe Fc fusion protein of antibody No. C1, C1-ld-Fc. C1-ld-Fc has a muchhigher coloration than the other two due to the long reaction time, andthus it shows better EC50. However, there is actually no difference inthe affinity of the three proteins for CTLA4.

5.2 Blocking Effect of CTLA4 Single-Domain Antibody Fc Fusion Protein toCTLA4-CD80 Interaction (by Competitive ELISA)

Plates were coated with 0.5 μg/well CTLA4-Fc fusion protein overnight at4° C. followed by the addition of 100 uL of a gradient dilution series(containing 250 ng/mL CD80-Fc-Biotin) of CTLA4 single-domain Fc fusionprotein obtained by Example 4.1 or 10D1 protein obtained by Example 4.2per well, allowed to react for 1 hour at room temperature. SA-HRP(purchased from Sigma) was added and allowed to react for 1 hour at roomtemperature. After adding chromogenic solution, absorbance was read at405 nm wavelength.

SotfMax Pro v5.4 was used for data processing and graphical analysis.Through four-parameter fitting, blocking curve and IC50 value of theantibody to CTLA4-CD80 were obtained. The results are shown in FIG. 7Aand FIG. 7B. It can be seen that the two different single-domainantibodies No. C27 and C1, whether humanized or the original sequence,have comparable ability to block the CTLA4-CD80 interaction, and aresuperior to the already marketed antibody of BMS (labelled as 10D1).

5.3 Binding Ability of Tetravalent CTLA4 Single-Domain Antibody FcFusion Protein to CTLA4 (by ELISA)

Plates were coated with CTLA4 single-domain Fc fusion protein obtainedby Example 4.1 and tetravalent CTLA4 single-domain antibody Fc fusionprotein obtained by Example 4.4 0.5 μg/well overnight at 4° C. followedby the addition of a gradient dilution series of CTLA4-Fc-Biotin,allowed to react for 1 hour at room temperature. SA-HRP (purchased fromSigma) was added and allowed to react for 1.5 hour at room temperature.After adding chromogenic solution, absorbance was read at 405 nmwavelength.

SotfMax Pro v5.4 was used for data processing and graphical analysis.Through four-parameter fitting, binding curve and EC50 value of theantibody to CTLA4 (all test antibody EC50 value of about 25-35 ng/mL)were obtained to reflect the affinity to CTLA4.

The results are shown in FIG. 8, where the longitudinal coordinate isOD405 and the horizontal ordinate is the concentration of CTLA4 singledomain antibody Fc-fusion protein (in ng/mL); triangle representstetravalent CTLA4 single-domain antibody Fc fusion protein,huC1v4-tet-Fc (SEQ ID NO: 128); square represents the humanized form Fcfusion protein of the antibody No. C1, huC1v4-Fc; circle represents Fcfusion protein of CTLA4 single-domain antibody No. C1, C1-ld-Fc. TheEC50 of the three proteins is slightly different, but considering thatthe molecular weight of the tetravalent protein is about 5/4 of thedivalent molecule, there is no difference in the affinity of the threeproteins for CTLA4 when converted into a molar concentration.

5.4 Identification and Comparison of the Binding Ability of Bivalent andTetravalent CTLA4 Single-Domain Antibody Fc Fusion Protein to CTLA4 (SPRMethod)

The binding kinetics of the CTLA4 single domain antibody Fc fusionprotein obtained in the above examples to recombinant human CTLA4 wasmeasured by the surface plasmon resonance (SRP) method using a BIAcoreX100 instrument. Recombinant camel anti human Fc antibody was coupled toa CM5 biosensor chip to obtain approximately 1000 response units (RU).For kinetic measurements, the antibodies were diluted (1.37 to 1000 nm)with HBS-EP+1× buffer (GE, cat #BR-1006-69) to fixed concentration andinjected for 120 s at 25° C. to ensure obtaining response value higherthan 100RU, and followed by serial three-times dilution for the fusionprotein of CTLA4 and mouse Fc and injected for 120 s at 25° C. with adissociation time of 30 min, regenerated with 10 mM Glycine-HCl (pH 2.0)for 120 s. Binding rates (kon) and dissociation rates (koff) werecalculated using a simple one-to-one Languir binding model (BIAcoreEvaluation Software version 3.2). The equilibrium dissociation constant(kD) is calculated as the ratio koff/kon.

The measured binding affinities of anti-CTLA4 antibodies are shown inTable 5. The results show that the affinity of the tetravalent moleculeis slightly lower than that of the divalent molecule, possibly due to acertain steric hindrance.

TABLE 5 Antibody Ka Kd KD HuC1V4-tet-Fc 1.318E+5 2.510E−5 1.905E−10HuC1V4-Ld-Fc 2.335E+5 2.035E−4 8.715E−115.4 Blocking Ability of Bivalent and Tetravalent CTLA4 Single Domain FcFusion Protein on CTLA4/CD80 Interaction (by Cell NeutralizationExperiment)

96-well plate was inoculated with 1.5×10⁵ Jurkat T cells (from theShanghai Cell Bank of Chinese Academy of Sciences), supplemented withAnti-human CD3 (50 ng/mL), and incubated at 37° C. for 15 min, then agradient dilution of the bivalent or tetravalent CTLA4 single-domainantibody Fc fusion protein (30 ng/ml-0.94 ng/ml, 100 ng/mL CTLA4-Fcfusion protein was added to the dilution) and equal-density Raji cells(from the Shanghai Cell Bank of Chinese Academy of Sciences) were added.After 24 hours of culture, the supernatant was collected for detectionof IL-2 expression. The data were processed with Soft Max to calculatethe effect of inhibiting IL-2 by the bivalent or tetravalent CTLA4single-domain antibody Fc fusion protein through neutralization ofCTLA4-Fc fusion protein. The effects were compared using EC50.

Results are shown in FIG. 9. The bivalent and tetravalent CTLA4single-domain antibody Fc fusion proteins (represented by square andtriangle, respectively) were equivalent in inhibiting CTLA4-CD80, andwere superior to the already marketed CTLA4 antibody of BMS (Labeled as10D1, indicated by a circle).

5.5 Binding Specificity of the CTLA4 Single Domain Antibody Fc FusionProtein for CTLA4 Protein

Human HEK293 cells transiently express human CTLA4, CD28, PD1 protein onmembranes by transient transfection of a plasmid carrying thefull-length human B7 family protein genes (pCDNA4, Invitrogen, CatV86220). The plasmid also allows the C-terminus of the target protein tobe fused to the EGFP protein so that the level of B7 family proteinexpressed on the membrane can be examined by green fluorescenceintensity. The constructed transient transfected cell lines include293-CTLA4-EGFP, 293-PD1-EGFP, 293-CD28-EGFP.

The constructed cells were resuspended in 0.5% PBS-BSA buffer andhuC1v4-tet-Fc antibody was added. At the same time, a negative controlof 2 μg of a single domain antibody against other unrelated target wasset up and incubated on ice for 20 minutes. After washing, eBiosciencesecondary antibody anti-hIg-PE was added, on ice for 20 min. Afterwashing, the cells were resuspended in 500 μl of 0.5% PBS-BSA buffer anddetected by flow cytometry.

The results are shown in FIG. 10. The upper row shows the control group,the lower row shows the sample groups. It is clear that huC1v4-tet-Fcspecifically binds to human CTLA4 protein only, not to other B7 familyproteins.

5.6 Binding of Tetravalent CTLA4 Single Domain Fc Fusion Protein toMonkey CTLA4 Protein

Monkey CTLA4-Fc protein was purchased from Yiqiao Shenzhou. Thebiotinylated protein huC1v4-tet-Fc-Biotin was obtained using the ThermoBiotinlytion kit with the tetravalent CTLA4 single domain antibody Fcfusion protein obtained in Example 4.4.

Plates were coated with monkey CTLA4-Fc protein or human CTLA4-Fcprotein, 0.5 μg/well, overnight at 4° C., followed by addition ofgradient dilution series of the huC1v4-tet-Fc-Biotin, allowed to reactfor 1 hour at room temperature. Then, SA-HRP (purchased from Sigma) wasadded and allowed to react at room temperature for 1.5 hour. Then,chromogenic agent was added and the absorbance was read at 405 nm.

SotfMax Pro v5.4 was used for data processing and graphical analysis.Through four-parameter fitting, blocking curve and EC50 value of theantibody to monkey CTLA4 or human CTLA4 were obtained.

The results are shown in FIG. 11, where longitudinal coordinate is OD405and the abscissa is the concentration of the tetravalent CTLA4 singledomain antibody Fc fusion protein (in ng/mL); the triangle representsthe binding with monkey CTLA4, the square and circle represent thebinding with human CTLA4. It can be seen that the tetravalent CTLA4single domain antibody Fc fusion protein can efficiently bind to monkeyCTLA4 protein.

5.7 Activation of PBMC by CTLA4 Single Domain Antibody Fc Fusion Protein

Peripheral blood mononuclear cells (PBMCs) were isolated from peripheralblood of healthy donors by density gradient centrifugation usingisolation solution for human lymphocytes (Tianjin Hao Yang).

Plates were coated with 0.3 μg/well anti-CD3 antibody overnight at 4° C.The next day, 1×10⁵ PBMCs were added to each well, at the same time, 10μg/mL CTLA4 single domain antibody Fc fusion protein huC1v4-Fc, huC27-Fcor the BMS CTLA4 antibody (named 10D1) were added to each wellrespectively. After cultured for 5 days, the supernatant was taken andthe level of IFN-γ in the supernatant was detected by IFN-γ ELISA kit(ebioscience).

The results are shown in FIG. 12. It can be seen that at theconcentration of 10 μg/mL, CTLA4 single domain antibody Fc fusionprotein combined with anti-CD3 antibody can enhance the secretion ofγ-interferon by PBMC cells, i.e. CTLA4 single domain antibody Fc fusionprotein enhances the activation of PBMC cells. Moreover, bothhuC1v4-Ld-Fc and huC27v3-Ld-Fc exhibited better activity than the BMSanti-CTLA4 antibody.

5.8 Activation of PBMC by Bivalent and Tetravalent CTLA4 Single DomainAntibody Fc Fusion Protein

Peripheral blood mononuclear cells (PBMCs) were isolated from peripheralblood of healthy donors by density gradient centrifugation usingisolation solution for human lymphocytes (Tianjin Hao Yang).

Plates were coated with 0.3 μg/well anti-CD3 antibody overnight at 4° C.The next day, 1×10⁵ PBMCs were added to each well, at the same time,0.03 ug/mL bivalent or tetravalent CTLA4 single domain antibody Fcfusion protein huC1v4-LD-Fc (labeled as Ld in the figure), huC1v4-tet-Fc(labeled as tet in figure) or the BMS CTLA4 antibody (named 10D1) wereadded to each well respectively. After cultured for 5 days, thesupernatant was taken and the level of IFN-γ in the supernatant wasdetected by IFN-γ ELISA kit (ebioscience).

The results are shown in FIG. 13. It can be seen that at the low doseconcentration of 0.03 μg/mL, CTLA4 single domain antibody Fc fusionprotein (both bivalent or tetravalent) combined with anti-CD3 antibodycan enhance the secretion of γ-interferon by PBMC cells, i.e. bivalentor tetravalent CTLA4 single domain antibody Fc fusion proteinsignificantly enhances the activation of PBMC cells at the low doseconcentration of 0.03 μg/mL. Moreover, tetravalent CTLA4 single domainantibody Fc fusion protein huC1v4-tet-Fc exhibited activity betteractivity than the BMS anti-CTLA4 antibody.

5.9 Activation of CD4+ T Cells by CTLA4 Single-Domain Antibody Fc FusionProtein in Dendritic Cell-T Cell Mixed Lymphoid Reaction

Peripheral blood mononuclear cells (PBMCs) were isolated from whiteblood cells of peripheral blood from healthy donors by density gradientcentrifugation using isolation solution for human lymphocytes (TianjinHao Yang). They were then incubated with serum-free RPMI 1640 medium for1-2 hours to remove non-adherent cells and cells were cultured in RPMIcontaining 10% FBS, 10 ng/ml GM-CSF and 20 ng/mL IL-4. After culturingfor 5-6 days, 10 ng/ml of TNF-α was added and incubated for 24 hours toobtain mature dendritic cells.

Dendritic cells obtained by this method were resuspended in RPMIcomplete medium, 2×10⁵/ml. Then 50 μl per well was added to a 96-wellU-bottom plate (Costar: 3799) and cultured in an incubator.

CD4+ T cells were isolated from PBMC of another donor using a magneticbead isolation kit (Miltenyi Biotec: 130-096-533) following theinstructions of the manufacturer.

1×10⁴ dendritic cells and 1×10⁵ CD4+ T cells obtained by the abovemethods were mixed, resuspended in RPMI complete medium and added to a96-well culture plate, and 50 μl of the cell mixture was added to eachwell. 100 μl per well of huC1v4-Ld-Fc diluted in RPMI complete mediumwas added to a final antibody concentration of 0.1 μg/ml or 0.01 μg/ml.Supernatants were collected 5-7 days after culture, and IFN-γ level inthe supernatant was detected by IFN-γ ELISA kit (ebioscience).

The results are shown in FIG. 14. It can be seen that CTLA4single-domain antibody Fc fusion protein can enhance the IFN-γ secretionof CD4+ T cells in mixed lymphocyte reaction. That is, the CTLA4blocking single-domain antibody Fc fusion protein enhances T cellactivation. Moreover, this biological activity wasconcentration-dependent, and a significant activation of T cells wasobserved at a low dose concentration (0.01 μg/ml).

5.10 Anti-Tumor Effect of Tetravalent CTLA4 Single Domain Antibody FcFusion Protein in Humanized Mice

CTLA4 humanized mice (i.e., mice expressing human CTLA4 protein) wereinoculated with 5×10⁵ MC38 tumor cells.

On day 7, 10, 13, and 16 post inoculation, 100 μg of the test sample orthe same amount of human immunoglobulin (as a control group) wasintraperitoneally administered. Tumor sizes were measured every two daysfrom day 5 post inoculation until day 20. The samples examined includedthe tetravalent CTLA4 single domain antibody Fc fusion protein (Tet inthe figure) and the CTLA4 monoclonal antibody, ipilimumab, which hasbeen marketed by BMS.

From the results shown in the figure below, the anti-tumor effect of thetetravalent CTLA4 single domain antibody Fc fusion protein in CTLA4humanized mice was comparable to that of BMS ipilimumab at a dose ofabout 5 mg/kg.

Example 6: Pharmacokinetics of CTLA4 Single Domain Antibody Fc FusionProtein in Rats

CTLA4 single domain antibody Fc fusion protein was single intravenously(IV) administrated to SD rats, the blood sample was collected atdifferent time points, and Elisa assay was used to determine theconcentration of the test substance in the plasma of the rats afteradministration of the test substance. The pharmacokinetic parameterswere calculated.

Animal selection: SD rats aged 6-8 weeks and weighed 200-300 g wereselected. They were randomly assigned to two groups, 8 rats each group,half male and half female.

Administration: Single intravenous (IV) administration at a dose of 10mg/kg.

Blood collection: Before administration, immediately afteradministration, at various time points after administration, about 0.5ml of blood was collected each time from the jugular vein of each rat.The collected blood was quickly centrifuged to separate the serum whichwas stored at −80° C. until analysis.

Blood drug concentration detection: The content of CTLA4 single domainantibody Fc fusion protein in serum was detected by sandwich ELISA. Theplate was coated with recombinant human CTLA4 protein to captureCTLA4-specific antibodies, and the Fc region was detected with goatanti-human IgG (Fc specific)-HRP antibody (Sigma) to ensure thedetection of intact CTLA4 single domain antibody Fc fusion protein.

Data processing: relevant pharmacokinetic parameters were calculatedusing blood drug concentration versus time curve, including AUC (0-t),AUC (0-∞), Cmax, Tmax, T1/2, Vss, MRT, and so on.

The curves of blood drug concentration versus time of the bivalent andtetravalent CTLA4 single domain antibody Fc fusion protein in rats areshown in FIG. 16 (a is the bivalent antibody huC1v4-Ld-Fc, and b is thetetravalent antibody huC1v4-tet-Fc). The pharmacokinetic parameters areshown in FIG. 17. The results showed that both the bivalent ortetravalent CTLA4 single domain antibody Fc fusion protein had a longerin vivo half-life in rats (more than 5 days), indicating good in vivostability. At the same time, in the case of maintaining the highestblood concentration, the tetravalent antibody had an in vivo half-lifethat is doubled (more than 11 days). It is thus concluded that themaintenance of the effective blood concentration of the tetravalentantibody in vivo is longer, and it is expected that the clinicaladministration can have a longer interval.

Example 7: Evaluation of the Druggability of CTLA4 Single DomainAntibody Fc Fusion Protein

7.1 Physicochemical Properties of CTLA4 Single Domain Antibody Fc FusionProtein

The affinity purified tetravalent CTLA4 single domain antibody Fc fusionprotein expressed by human 293HEK cells was obtained by the methoddescribed in Example 4.4. Then its druggability was assessed bypreliminary physical and chemical properties analysis through SE-HPLC,CE under reducing conditions, CE, WCX, DSC under non-reducingconditions. The specific values are set forth in the following table.From these data, it can be preliminarily determined that the tetravalentCTLA4 single domain antibody Fc fusion protein has good physical andchemical properties and is suitable for industrial scale production.

TABLE 6 DSC(single- SE-HPLC domain Expression SE-HPLC polymer antibody,CE CE Protein Name Level purity (%) content (%) Tm, □) reduction %non-reduction % Deamination % HuC1v4-tet-Fc ~400 mg/L 97.6 2.0 71.5 99.397.9 77.2 Thermal Destruction Test of CTLA4 Single Domain Antibody Fc FusionProtein

The CTLA4 single domain antibody Fc fusion protein was concentrated byUF/DF and exchanged into PBS buffer to prepare a 20 mg/mL solution, andthe thermal destruction test was performed with accelerating at 40° C.for 30 days. Samples of day 0, 10, 20, and 30 were tested for purity bySE-HPLC, and the change trend is shown in the following figure. It canbe seen that at 40° C., without optimization of the preparation,although the purity of the main peak was slightly decreased, no obvioustendency of aggregation or degradation was observed. At the same time,considering its higher Tm value, it can be determined that the proteinhas good thermal stability.

Example 8: Evaluation of Preliminary Toxicity of CTLA4 Single DomainAntibody Fc Fusion Protein

Four cynomolgus monkeys were randomly divided into two groups, two ineach group, half male and half female, as the low and high dose groupsrespectively (7.5 and 30 mg/kg, respectively). Tetravalent CTLA4 singledomain antibody Fc fusion protein was administered once via a posteriorlimb vein bolus. Before administration (D-1), D15 and D29 postadministration, the indexes of body weight, blood cell count, bloodcoagulation function, blood biochemistry and immunogenicity weredetected.

During the test, four animals showed no death or impending death. As forcynomolgus monkeys received single dose 7.5-30 mpk, significantlyincreases of hematological indexes lymph, Eos, Baso, Mono were observed.Clinical observation, weight, coagulation function, blood biochemicalindicators of other groups of animals did not show changes withtoxicological significance.

Thus, a single intravenous injection to cynomolgus monkeys at 7.5 and 30mpk showed no significant toxicity, with NOAEL greater than or equal to30 mg/kg. The non-clinical NOAEL reported for ipilimumab of BMS isapproximately 10 mg/kg. It can be determined that the CTLA4 singledomain antibody Fc fusion protein has lower toxic side effects thanipilimumab.

What is claimed is:
 1. A CTLA4-binding protein, which can specificallybind to CTLA4 and comprises at least one immunoglobulin single variabledomain, said immunoglobulin single variable domain comprises CDR1comprising the amino acid sequence of SEQ ID NO:25, CDR2 comprising theamino acid sequence of SEQ ID NO:26, CDR3 comprising the amino acidsequence of SEQ ID NO:27.
 2. The CTLA4-binding protein of claim 1,wherein said immunoglobulin single variable domain is a VHH.
 3. TheCTLA4-binding protein of claim 2, wherein the VHH is a humanized VHH. 4.The CTLA4-binding protein of claim 3, wherein said VHH comprises anamino acid sequence having at least 80%, or at least 90%, or at least95%, or at least 99% sequence identity to SEQ ID NO:84.
 5. TheCTLA4-binding protein of claim 2, wherein said VHH comprise the aminoacid sequence of SEQ ID NO:84.
 6. The CTLA4-binding protein of claim 1,which comprises two said immunoglobulin single variable domain.
 7. TheCTLA4-binding protein of claim 1, which further comprises animmunoglobulin Fc region.
 8. The CTLA4-binding protein of claim 7, whichcomprise the amino acid sequence of SEQ ID NO:
 114. 9. The CTLA4-bindingprotein of claim 8, wherein the immunoglobulin Fc region is an Fc regionof human immunoglobulin, preferably an Fc region of human IgG1.
 10. TheCTLA4-binding protein of claim 9, wherein the amino acid sequence of theimmunoglobulin Fc region is SEQ ID NO:132.
 11. The CTLA4-binding proteinof claim 1 or 4, which has at least one of the following features: (a)binding to human CTLA4 with a KD of less than 1×10⁻⁷ M; (b) blocking theinteraction of CTLA4 with CD80 and/or CD86; (c) enhancing activation ofPBMCs and/or T cells; (d) inhibiting tumor growth.
 12. The CTLA4-bindingprotein of claim 1 or 4, which binds to CTLA4 with a KD of less than1×10⁻⁷ M, preferably less than 1×10⁻⁸ M, more preferably less than1×10⁻⁹ M, more preferably less than 1×10⁻¹⁰ M.